Transplantation of bone marrow stromal cells for treatment of neurodegenerative diseases

ABSTRACT

The present invention relates to a treatment of an autoimmune demyelinating disease/disorder. Also included in the present invention is the use of bone marrow stromal cells for the treatment of multiple sclerosis (MS).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 11/027,881, filed Dec. 30, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 09/980,614,filed on Apr. 17, 2002, which is a national phase application filedunder 35 U.S.C. §371, claiming the benefit of priority of InternationalApplication No. PCT/US00/12875, filed May 11, 2000, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/134,344, filed May 14, 1999, all of which are herein incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Most central nervous system (CNS) injuries include stroke, trauma,hypoxia-ischemia, multiple sclerosis, seizure, infection, and poisoningdirectly or indirectly involve a disruption of blood supply to the CNS.These injuries share the same common pathologic process of rapidcerebral edema leading to irreversible brain damage and eventually tobrain cell death.

One common injury to the CNS is stroke which is the destruction of braintissue as a result of intracerebral hemorrhage or ischemia. Stroke maybe caused by reduced blood flow or ischemia that results in deficientblood supply and death of tissues in one area of the brain (infarction).The causes of ischemic stroke include blood clots that form in the bloodvessels in the brain (thrombus) and blood clots or pieces ofatherosclerotic plaque or other material that travel to the brain fromanother location (emboli). Bleeding (hemorrhage) within the brain mayalso cause symptoms that mimic stroke.

The CNS tissue is highly dependent on blood supply and is veryvulnerable to interruption of blood supply. Without neuroprotection,even a brief interruption of the blood flow to the CNS can causeneurological deficit. The brain is believed to tolerate completeinterruption of blood flow for a maximum of about 5 to 10 minutes. Ithas been observed that after blood flow is restored to areas of thebrain that have suffered an ischemic injury, secondary hemodynamicdisturbances have long lasting effects that interfere with the abilityof the blood to supply oxygen to CNS tissues. Similarly, interruption ofthe blood flow to the spinal cord, for even short periods of time, canresult in paralysis.

Recognition of the “ischemic penumbra,” a region of reduced cerebralblood flow in which cell death might be prevented, has focused attentionon treatments that might minimize or reverse brain damage when thetreatments are administered soon after stroke onset. To date, severalclasses of neuroprotective compounds have been investigated for acutestroke. They have included calcium channel antagonists,N-methyl-D-aspartate (NMDA) receptor antagonists, free radicalscavengers, anti-intercellular adhesion molecule 1 antibody, GM-1ganglioside, γ-aminobutyric acid agonists, and sodium channelantagonists, among others. Results from various trials have yieldeddisappointing efficacy results and some evidence of safety problems,including increased mortality or psychotic effects which resulted intheir early termination.

Multiple sclerosis (MS) is another disease of the CNS. MS is aninflammatory demyelinating disease, which typically displays arelapsing-remitting course characterized by episodes of neurologicaldisability followed by periods of partial or complete clinical remission(Lucchinetti et al., 2000, Ann. Neurol. 47:707-717; Hemmer et al., 2002,Nat. Rev. Neurosci. 3:291-301). Most patients later enter a progressivephase of steady decline of neurological function. Severe axonal loss andneuronal death are frequent in MS (Ferguson et al., 1997, Brain 120 (Pt.3):393-399; Trapp et al., 1998, N. Engl. J. Med 338:278-285; Peterson etal., 2001, Ann. Neurol. 50:389-400; Bjartmar et al., 2003, Neurotox.Res. 5:157-164). Axonal loss is a major cause of permanent neurologicaldeficit in MS (Wujek et al., 2002, J. Neuropathol. Exp. Neurol.61:23-32; Bjartmar et al., 2003, J. Neurol. Sci. 206:165-171; Medana etal., 2003, Brain 126:515-530). Chronically demyelinated axons maydegenerate due to a lack of myelin-derived trophic support (Bjartmar etal., 2003, J. Neurol. Sci. 206:165-171); however, no current therapiesfor MS are known provide at axonal protection (Bectold, et al., 2004,Ann. Neurol. 55:607-616).

Cellular therapy serves as an alternative to drug therapy. It has beendemonstrated that intracerebral transplantation of donor cells fromembryonic tissue may promote neurogenesis (Snyder et al., 1997 AdvNeurol. 72:121-32). Intrastriatal fetal graft has been used toreconstruct damaged basal ganglia circuits and to ameliorate behavioraldeficits in a mammalian model of ischemia (Goto et al., 1997 Exp Neurol.147:503-9). Fetal hematopoietic stem cells (HSCs) transplanted into theadult organism or adult HSCs transplanted into an embryo results in achimera that reflects the endogenous cells within the microenvironmentinto which the cells were seeded (Geiger et al., 1998, Immunol Today19:236-41). Pluripotent stem cells are harbored in the adult CNS and theadult brain can form new neurons (Gage, 1998 Curr. Opin. Neurobiol.8:671-6; Kempermann and Gage, 1998 Nat Med. 4:555-7).

Bone marrow contains at least two types of stem cells, hematopoieticstem cells and stem cells of non-hematopoietic tissues variouslyreferred to as mesenchymal stem cells or marrow stromal cells (MSCs) orbone marrow stromal cells (BMSCs). These terms are used synonymouslythroughout herein. MSCs are of interest because they are easily isolatedfrom a small aspirate of bone marrow and they readily generatesingle-cell derived colonies. The single-cell derived colonies can beexpanded through as many as 50 population doublings in about 10 weeks,and can differentiate into osteoblasts, adipocytes, chondrocytes(Friedenstein et al., 1970 Cell Tissue Kinet. 3:393-403;Castro-Malaspina et al., 1980 Blood 56:289-301; Beresford et al., 1992J. Cell Sci. 102:341-351; Prockop, 1997 Science 276:71-74), myocytes(Wakitani et al., 1995 Muscle Nerve 18:1417-1426), astrocytes,oligodendrocytes, and neurons (Azizi et al., 1998 Proc. Natl. Acad. Sci.USA 95:3908-3913); Kopen et al., 1999 Proc. Natl. Acad. Sci. USA96:10711-10716; Chopp et al., 2000 Neuroreport II 3001-3005; Woodbury etal., 2000 Neuroscience Res. 61:364-370). For these reasons, MSCs arecurrently being tested for their potential use in cell and gene therapyof a number of human diseases (Horwitz et al., 1999 Nat. Med. 5:309-313;Caplan, et al. 2000 Clin. Orthoped. 379:567-570).

MSCs constitute an alternative source of pluripotent stem cells. Underphysiological conditions they maintain the architecture of bone marrowand regulate hematopoiesis with the help of different cell adhesionmolecules and the secretion of cytokines, respectively (Clark andKeating, 1995 Ann NY Acad Sci 770:70-78). MSCs grown out of bone marrowby their selective attachment to tissue culture plastic can beefficiently expanded (Azizi et al., 1998 Proc Natl Acad Sci USA95:3908-3913; Colter et al., 2000 Proc Natl Acad Sci USA 97:3213-218)and genetically manipulated (Schwarz et al. 1999 Hum Gene Ther10:2539-2549).

MSC are also referred to as mesenchymal stem cells because they arecapable of differentiating into multiple mesodermal tissues, includingbone (Beresford et al., 1992 J Cell Sci 102:341-351), cartilage (Lennonet al., 1995 Exp Cell Res 219:211-222), fat (Beresford et al., 1992 J.Cell. Sci. 102:341-351) and muscle (Wakitani et al., 1995 Muscle Nerve18:1417-1426). In addition, differentiation into neuron-like cellsexpressing neuronal markers has been reported (Woodbury et al., 2000 JNeurosci Res 61:364-370; Sanchez-Ramos et al., 2000 Exp Neurol164:247-256; Deng et al., 2001 Biochem Biophys Res Commun 282:148-152),suggesting that MSC may be capable of overcoming germ layer commitment.

The concept of transplantation of bone marrow has been studied byothers. For example, in the Azizi et al. reference, the investigatorstransplanted human bone marrow stromal cells (hBMSCs) into the brains ofalbino rats (Azizi et al., 1998 Proc Natl Acad Sci USA 95:3908-3913).Their primary observations were that hBMSCs can engraft, migrate andsurvive in a manner similar to rat astrocytes. Further, it has beendemonstrated that the bone marrow cells when implanted into the brain ofadult mice can differentiate into microglia and macroglia (Eglitis etal., Proc Natl Acad Sci USA 1997 94:4080-5). Again, this occurred whenthe bone marrow cells were transplanted into the brain of normal mice.There have been many attempts made to use bone marrow stromal cells incell therapy in an animal model. However, there has been little evidenceof using bone marrow stromal cells in a diseased animal model orotherwise an animal that is suffering from a disease. Thus, there is along felt need in the art for efficient and directed means of treating aneurodegenerative disease such as MS in a mammal. The present inventionsatisfies this need.

BRIEF SUMMARY OF THE INVENTION

According to the present invention, there is provided a method oftreating a mammal suffering from a central nervous system (CNS) injuryand/or a neurodegenerative disease. The method includes the steps ofculturing bone marrow stromal cells and transplanting or otherwiseadministering the bone marrow stromal cells into the brain of a mammalin need thereof. In addition, the present invention encompasses acomposition comprising bone marrow cells and embryonic brain tissue forthe use in the treatment of CNS injury and/or neurodegeneration.

Also provided is a method of activating the differentiation of neuralcells in an injured brain comprising the steps of transplanting bonemarrow stromal cells adjacent to the injured brain cells by way ofintravascular (intraarterial, intravenous) administration of the bonemarrow stromal cells to the mammal and having the bone marrow stromalcells activate the endogenous central nervous system stem cells todifferentiate into neurons.

In another aspect, the invention includes a method of stimulating brainparenchymal cells to express an array of trophic factors including butnot limited to NGF, BDNF, VEGF, and bFGF. The method comprises the stepsof transplanting bone marrow stromal cells adjacent to the injured braincells by way of intravascular (intraarterial or intravenous)administration of the bone marrow stromal cells to the mammal. Theexpression of neurothrophic factors by parenchymal cells stimulated byMSCs provides a therapeutic benefit.

A method of treating injured and degenerative brain using the cells ofthe present invention is also provided. The method comprises the stepsof preparing bone marrow stromal cells and transplanting bone marrowstromal cells near the injured brain cells by way of intravascularadministration of the cells.

In addition to using bone marrow stromal cells, whole bone marrow andcellular components of bone marrow have been employed (i.e. mesenchymalstem cells (MSCs); hematopoietic stem cells (HSCs) to treat stroke andtraumatic brain injury. Cellular components of bone marrow were culturedin a special medium and in medium comprising neurotrophins (i.e. NerveGrowth Factor (NGF), Brain-derived neurotrophic factor (BDNF)). Cellswere injected either directly into the brain, into the internal carotidartery or into a femoral vein. The outcome of having the cellsadministered into the brain were measured using double stainingimmunohistochemistry techniques to morphologically identify phenotypictransformation of bone marrow cells, and behavioral and functional teststo identify neurological deficits of the mammal. The data presentedherein demonstrate that treatment of among others, stroke, spinal cordinjury, or traumatic brain injury with whole bone marrow or cellularcomponents significantly reduces functional deficits. Bone marrow cellsalso express phenotypes of parenchymal cells.

In addition, mice treated with the neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce symptomsof Parkinson's disease, were treated with bone marrow cells or bonemarrow stromal cells by delivering the cells by the route, including butnot limited to, intracerebral and intravascular delivery of the cells tothe mammal. Parkinson's symptoms were significantly reduced in micetreated with either bone marrow cells or bone marrow stromal cells.These data demonstrate that these cells can be employed to treat neuralinjury and neurodegenerative disease.

Furthermore, a mouse experimental autoimmune encephalomyelitis (EAE)model which mimics multiple sclerosis (MS) was employed to demonstratethat administration of bone marrow stromal cell to a mammal in needthereof can treat a neurodegenerative disease associated withdemyelination. Accordingly, the invention includes a method of treatinga neurodegenerative disease associated with inflammatory demyelinationusing bone marrow stromal cells. Preferably, the neurodegenerativedisease is MS.

Also encompassed in the present invention is a composition comprising anaggregate, composed of neural stem cells from the fetal neurosphere,MSCs from adult bone marrow and cerebro-spinal fluid from adult Wistarrats (called NMCspheres). These NMCspheres have been successfully usedto treat stroke and brain trauma, and can be employed to treatneurodegenerative disease.

Accordingly, the present invention encompasses methods and compositionsfor the culturing of bone marrow stromal cells in neurotrophins, and theintraparenchymal and intravascular administration of these cells(cultured in the presence or absence of a growth factor), for therapyand the treatment of stroke, trauma and Parkinson's disease using bonemarrow. In addition, the cells can be used to treat a neurodegenerativedisease associated with demyelination.

The invention relates to a method of treating a mammal having a disease,disorder or condition of the CNS. The method comprises obtaining a bonemarrow sample from donor, isolating a stromal cell from the bone marrowsample, and administering the isolated stromal cell to the CNS of themammal, wherein the presence of the isolated stromal cell in the CNSeffects treatment of the disease, disorder or condition.

In one aspect, the presence of the isolated stromal cell in the CNS ofthe mammal induces angiogenesis. In another aspect, the presence of theisolated stromal cell in the CNS of the mammal induces neurogenesis. Inyet another aspect, the presence of the isolated stromal cell in the CNSof the mammal induces synaptogenesis.

In one aspect, the presence of the isolated stromal cell in the CNS ofthe mammal does not induce an immune response against the stromal cell.

In another aspect, the mammal is a human.

In another aspect, the donor is a human who is not suffering from adisease, disorder or condition of the central nervous system.

In yet another aspect, the human donor is allogeneic, syngeneic orxenogeneic with the human patient.

In a further aspect, the human donor is the human patient.

In one aspect, the disease, disorder or condition of the CNS is selectedfrom the group consisting of a genetic disease, an ischemic inducedinjury, a spinal cord injury, stroke and Parkinson's disease.

In yet another aspect, the disease, disorder or condition of the CNS isan inflammatory demyelinating disease. More preferably, the disease,disorder or condition of the CNS is MS.

In another aspect, the disease, disorder or condition is injury to thetissues or cells of the CNS. In yet another aspect, the disease,disorder or condition is within the brain of the patient.

In a further aspect, the isolated stromal cell administered to the CNSremains present in the CNS. In another aspect, the isolated stromal celladministered to the CNS replicates in the CNS.

In yet another aspect, the stromal cell administered to the CNS does notresult in a cell replacement therapy. Preferably, the stromal cellinduces endogenous neighboring cells to express a growth factor. Morepreferably, the endogenous neighboring cell is a parenchymal or vascularcell.

In yet another aspect, prior to administering the isolated stromal cell,the cell is cultured in vitro.

In one aspect, the isolated stromal cell is administered to the mammalby a route selected from the group consisting of intravascular,intracerebral, parenteral, intraperitoneal, intravenous, epidural,intraspinal, intrastemal, intra-articular, intra-synovial, intrathecal,intra-arterial, intracardiac, and intramuscular.

In another aspect, the isolated stromal cell in the CNS of the mammalsecretes a factor selected from the group consisting of a growth factor,a trophic factor and a cytokine. In a further aspect, the secretedfactor is selected from the group consisting of leukemia inhibitoryfactor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growthfactor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6,glial-derived neurotrophic factor (GDNF), granulocyte colony-stimulatingfactor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-likegrowth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8,monocyte chemotactic protein (MCP-1), mononuclear phagocytecolony-stimulating factor (M-CSF), neurotrophic factors (NT3), tissueinhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor(TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinaseplasminogen activator receptor (uPAR), bone morphogenetic protein 4(BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromalcell-derived factor-1 (SDF-1), platelet derived growth factor-BB(PDGFBB), transforming growth factors beta (TGFβ-1) and TGFβ-3.

In yet a further aspect, prior to administering the isolated stromalcell, the isolated stromal cell is transfected with an isolated nucleicacid encoding a therapeutic protein, wherein when the protein issecreted by the stromal cell and the secreted protein serves to effecttreatment of said disease, disorder or condition.

The invention includes administering an isolated stromal cell to themammal at the site of injury.

In another aspect, the stromal cell is administered to the mammal at anadjacent site to the site of injury. In one aspect, followingadministering the stromal cell into the mammal, the stromal cellmigrates to the site of injury.

In a further aspect, the stromal cell present in the CNS activates theproliferation of an endogenous neighboring cell. In a further aspect,the endogenous neighboring cell is an astrocyte.

In one aspect, the stromal cell activates the MEK/Akt pathway inneighboring cells. In another aspect, the stromal cell activates thePI3K/Erk pathway in neighboring cells. In a further aspect, the stromalcell activates the differentiation of neighboring cells.

In yet another aspect, the stromal cell exhibits at least one markercharacteristic of a cell of the CNS. In further aspect, the marker isselected from the group consisting of class III β-tubulin, the M subunitof neurofiliments, tyrosine hydroxylase, gluatmate receptor subunits ofthe GluR1-4 and GluR6 classes, glial fibrillary acidic protein, myelinbasic protein, brain factor 1, NeuN, NF-M, NSE, nestin, and trkA.

The invention includes administering an isolated stromal cellconcomitantly with a growth factor to the mammal.

In one aspect, the stromal cell administered to the patient preventsaxonal fiber loss in the cells of the mammal.

In another aspect, the stromal cell administered to the patient preventsor reduces demyelination in the cells from the mammal.

The invention includes administering an isolated stromal cell to themammal in the absence of immunosupppressive agents.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIG. 1A through Figure B, is a diagram of the threeregions of the rat brain after two hours of middle cerebral arteryocclusion (MCAo) with bone marrow transplantation.

FIG. 2, comprising FIG. 2A through FIG. 2L is an image depicting bonemarrow cells in an H&E prepared section in the immunoreactivity ofrepresentative proteins in the (ischemic boundary zone) IBZ of a seriesof adjacent sections from rats sacrificed four days after bone marrowtransplantation (FIG. 2A through FIG. 2H). FIG. 2I depicts the neuronalspecific nuclear protein, NeuN. FIG. 2J demonstrates that the bonemarrow transplantation of the cells adjacent to the ependymal cellsexhibited reactivity for the neuronal marker, MAP-2; and FIG. 2K-FIG. 2Ldepict that the cells of the (subventricular zone) SVZ express Neuro Dand glial fibrillary acidic protein (GFAP) protein markers.

FIG. 3, comprising FIG. 3A through FIG. 3H, is a series of imagesdepicting H&E prepared sections of cerebral tissue after MCAo and havingbone marrow cells transplanted into the mammal after MCAo.

FIG. 3, comprising FIG. 3I through FIG. 3J, is an image depicting theTUNEL staining exhibiting apoptotic-like cells within the bone marrowgrafting at four days following transplantation.

FIG. 4, comprising FIG. 4A through FIG. 4C, depicts data from theadhesive-removal test, the rotarod-motor test and the NeurologicalSeverity Score (NSS), respectively.

FIG. 5, comprising FIG. 5A through FIG. 5B, depicts grafts demonstratingthat mice treated with transplanted MSCs exhibited a significantimprovement in the duration on the rotarod and an improved neurologicalfunction compared to vehicle treated mammals.

FIG. 6, comprising FIG. 6A through FIG. 6B, depicts that rats thatreceived MSC intraarterial transplantation exhibited significantimprovement on the adhesive-removal test and the modified NeurologicalSeverity Scores (mNSS) at 14 days following transplantation comparedwith control mammals.

FIG. 7, comprising FIG. 7A through FIG. 7B depicts functional data fromrats receiving administration of MSCs intravenously compared withcontrol-ischemia rats not receiving MSCs.

FIG. 8 depicts rotarod data from mice subjected to1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity;

FIG. 9, comprising FIG. 9A through FIG. 9D depicts the morphologicalchanges, i.e. most shrunk pigmented neurons disappeared and only few ofthem were observed in the substantia nigra at 45 days after MSCtransplantation in MPTP induced Parkinson's diseased (MPTP-PD) mice;viable 5-bromo-2-deoxyuridine (BrdU) immunoreactive cells identified inthe injected area and migrated to variable distances into the hoststriatum at 45 days after transplantation of the MSCs; double stainingshows that scattered BrdU reactive cells express tyrosine hydroxylase(TH) immunoreactivity within the grafts;

FIG. 10 depicts data from the Basso-Beaftie-Bresnahan (BBB) test frommammals subjected to spinal cord injury.

FIG. 11 is an image depicting the composite MSC neurosphere nine daysafter cell-neurosphere integration.

FIG. 12, comprising FIGS. 12A and 12B, is a series of imagesdemonstrating that treatment with hMSCs improves survival rate andneurological functional recovery in experimental autoimmuneencephalomyelitis (EAE) mice. FIG. 12A demonstrates that the survivalrates for hMSCs treated mice at weeks 10, 20, 35, and 45 weresignificantly higher than those in the PBS group (p<0.01). FIG. 12Bdemonstrates that functional scores were significantly lower among micetreated with hMSCs compared with PBS treated mice as early as 1 week upto 45 weeks (p<0.05).

FIG. 13, comprising FIGS. 13A through 13D, is a series of imagesdemonstrating that hMSC treatment increases axonal density in the whitematter of EAE brain. FIGS. 13A-13B and FIGS. 13C-13D depict reduced areaof axonal loss in the striatum and corpus callosum, respectively,between the hMSC treatment group compared with the PBS treatment group.

FIG. 14, comprising FIGS. 14A through 14C, is a series of imagesdemonstrating that administration of hMSCs increases NGF expression inthe CNS of EAE mice. FIG. 14A is an image depicting NGF cell expressionin the EAE brain treated with hMSCs or PBS. FIG. 14B is a graphdepicting increased numbers of NGF reactive cells in the brain at 1, 10,20, 35 and 45 weeks compared with the PBS treatment. FIG. 14C is animage depicting that about 50-70% of NGF⁺ cells co-localizes with NeuN⁺cells.

FIG. 15 is a graph demonstrating that hMSCs are present in EAE brainfollowing transplantation as measured by the presence of MAB1281 cells.

FIG. 16, comprising FIGS. 16A-16I, is a series of images depicting thephenotype of the transplanted hMSC. FIGS. 16D-16F demonstrates that lessthan about 5% of MAB1281⁺ cells co-localized with NG2⁺ cells. FIGS.16A-16C and 16C-16I demonstrate that about 10% of MAB1281⁺ cellsco-localized with GFAP⁺ cells and MAP-2⁺ cells, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating neural injury andneurodegeneration using transplantation of bone marrow stromal cells. Ithas been determined that bone marrow stromal cells present withininjured brain and/or spinal cord produce an array of factors including,but not limited to, cytokines and growth factors. The bone marrowstromal cells activate among others, endogenous stem cells and ependymalcells in the brain, to proliferate and differentiate into parenchymalcells including, but not limited to, neurons. These new neurons can bepresent at sites adjacent to the sites of injury. Thus, the bone marrowstromal cells activate endogenous CNS stem cells to differentiate intoamong others, neurons. The bone marrow stromal cells also producefactors including, but not limited to cytokines and growth factors, thatpromote repair and plasticity of the brain. In addition, the bone marrowstromal cells can induce angiogenesis.

In addition, the invention includes a method of transplanting bonemarrow stromal cells to a mammal in need thereof, such as a mammalhaving MS, where the bone marrow stromal cells induce expression ofgrowth factors within CNS cells. In one aspect, the bone marrow stromalcells stimulate brain parenchymal cells to express NGF. The expressionof NGF by the parenchymal cells provides an elevated level of NGFpresent in the CNS that otherwise would be at a lower level in theabsence of bone marrow stromal cells. The elevated level of NGF presentin the CNS provides a therapeutic benefit, including but not limited tostimulating axonal repair, prevent demyelination, reducing axonal loss,stimulating oligodendrocyte growth, stimulated oligodendrocytedifferentiation, enhancing survival of differentiated oligodendrocytes,and exhibiting immunomodulatory effects. Therefore, in some instances,the therapeutic effect from transplantation of bone marrow stromal cellis not due to cell replacement therapy where the transplanted bonemarrow stromal cells differentiate into neuronal cells for replacementof damaged endogenous neuronal cells, but rather the interaction withendogenous cells to induce endogenous cells to secrete growth factorssuch as NGF.

The present invention encompasses methods of culturing bone marrowstromal cells, and methods for administering the cells of the presentinvention to a mammal. The cells can be transplanted into the penumbraltissue, which is a tissue adjacent to a lesion. The tissue adjacent tothe lesion provides a receptive environment, similar to that of adevelopmental brain, for the survival and differentiation of the bonemarrow stromal cells. It is based on this activity that the bone marrowstromal cells are useful in treating neural injury and neurodegenerationwherein brain and/or spinal cord damage has occurred.

In addition, bone marrow stromal cells are effective in treating neuralinjury and degeneration when these cells are administeredintravascularly, i.e. intraarterially or intravenously. Therefore, aftersuch brain injury, when the brain tissue is damaged, in an effort tocompensate for the lost tissue, the administration of bone marrowstromal cells can provide a sufficient source of cells to promotecompensatory responses of the brain to such damage.

The cells of the present invention can be administered into including,but not limited to ischemic brain, injured brain, injured spinal cordand into brain that exhibits symptoms of Parkinson's disease. In someinstances, the cells are administered to a brain of a mammal thatexhibits symptoms of MS. In any event, transplantation of the cells intothe brain can also be performed with co-transplantation of growthfactors including, but not limited to brain derived neurotrophic factor(BDNF) and nerve growth factor (NGF). The cells of the invention arecultured with NGF prior to transplantation into a recipient.

Transplantation can be performed at various time points (i.e., from fourhours to two days after stroke, from one to seven days after trauma,seven days after spinal cord injury and fourteen days after initiationof Parkinson's disease) after experimental stroke in both the rat andthe mouse. The data presented herein indicate that the transplantationof bone marrow or components into ischemic brain results indifferentiation of the bone marrow cells into the brain parenchymalcells, including but not limited to neurons. In addition, endogenousbrain stem cells are activated to proliferate and differentiate intoparenchymal cells. The cells of the invention migrate to differentregions within the brain including, but not limited to, the hippocampus,the olfactory bulb and the cortex. There is also improved functionaloutcome in rats treated with bone marrow transplantation cultured withor in combination with growth factors. The disclosure hereindemonstrates that the cells of the invention can also be used to provideimproved functional outcomes in higher mammals, including but notlimited to humans.

The disclosure presented herein also indicates that the transplantationof bone marrow stromal cells into a brain of a mammal that exhibitscharacteristics of MS or otherwise a mammal suffering fromimmune-mediated demyelination, reduces axonal loss in the brain. Inaddition, the transplantation of bone marrow stromal cells contributesto expression of NGF from endogenous parenchymal cells. The secretion ofNGF by endogenous parenchymal provides both neurotrophic andimmunomodulatory effects.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

As used herein, the term “allogeneic” is meant to refer to any materialderived from a different mammal of the same species.

“Xenogeneic” refers to any material derived from a mammal of a differentspecies.

As used herein, the term “bone marrow stromal cells (BMSCs),” “stromalcells,” “mesenchymal stem cells” or “MSCs” are used interchangeably andrefer to the small fraction of cells in bone marrow which can serve asstem cell-like precursors to osteocytes, chondrocytes, and adipocytes.Bone marrow stromal cells have been studied extensively(Castro-Malaspina et al., 1980, Blood 56:289-30125; Piersma et al.,1985, Exp. Hematol 13:237-243; Simmons et al., 1991, Blood 78:55-62;Beresford et al., 1992, J. Cell. Sci. 102:341-3 51; Liesveld et al.,1989, Blood 73:1794-1800; Liesveld et al., Exp. Hematol 19:63-70;Bennett et al., 1991, J. Cell. Sci. 99:131-139). Bone marrow stromalcells may be derived from any animal. In some embodiments, stromal cellsare derived from humans.

“Differentiation medium” is used herein to refer to a cell growth mediumcomprising an additive or a lack of an additive such that a stem cell,embryonic stem cell, ES-like cell, MSCs, neurosphere, NSC or other suchprogenitor cell, that is not fully differentiated when incubated in themedium, develops into a cell with some or all of the characteristics ofa differentiated cell.

As used herein, the term “disease, disorder or condition of the centralnervous system” is meant to refer to a disease, disorder or a conditionwhich is caused by a genetic mutation in a gene that is expressed bycells of the central nervous system such that one of the effects of sucha mutation is manifested by abnormal structure and/or function of thecentral nervous system, such as, for example, neurodegenerative diseaseor primary tumor formation. Such genetic defects may be the result of amutated, non-functional or under-expressed gene in a cell of the centralnervous system. The term should also be construed to encompass otherpathologies in the central nervous system which are not the result of agenetic defect per se in cells of the central nervous system, but ratherare the result of infiltration of the central nervous system by cellswhich do not originate in the central nervous system, for example,metastatic tumor formation in the central nervous system. The termshould also be construed to include trauma to the central nervous systeminduced by direct injury to the tissues of the central nervous system.The term should also include a neurodegenerative disease associated withdemyelination of cells of the CNS. An example of such a disease ismultiple sclerosis (MS).

“Neural stem cell” or “NSC” is used herein to refer to undifferentiated,multipotent, self-renewing neural cell. A neural stem cell is aclonogenic multipotent stem cell which is able to divide and, underappropriate conditions, has self-renewal capability and can terminallydifferentiate into among others, neurons, astrocytes, andoligodendrocytes. Hence, the neural stem cell is “multipotent” becausestem cell progeny have multiple differentiation pathways. A neural stemcell is capable of self maintenance, meaning that with each celldivision, one daughter cell will also be, on average, a stem cell.

“Neurosphere” is used herein to refer to a neural stem cell/progenitorcell wherein Nestin expression can be detected, including, inter alia,by immunostaining to detect Nestin protein in the cell. Neurospheres areaggregates of proliferating neural stem and progenitor cells and theformation of neurosphere is a characteristic feature of neural stemcells in in vitro culture.

As used herein, “central nervous system” should be construed to includebrain and/or the spinal cord of a mammal. The term may also include theeye and optic nerve in some instances.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced from or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

The phrase “substantially homogeneous population of cells” as usedherein should be construed to mean a population of cells wherein atleast 75% of the cells exhibit the same phenotype.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered. Also, as used herein, a“therapeutically effective amount” is the amount of cells which issufficient to provide a beneficial effect to the subject to which thecells are administered.

Description

The present invention is based on the discovery that MSCs candifferentiate into neurons and other parenchymal cells. In addition, thepresent invention is based on the discovery that MSCs can secrete afactor including, but not limited to NGF, BDNF, VEGF, and bFGF, that isbeneficial to neighboring and/or distal cells. The disclosure hereindemonstrates that when MSCs are introduced into a mammal, the MSCsactivate endogenous cells to proliferate and differentiate. In someinstances, the MSCs induce endogenous cells to express NGF. Preferably,the endogenous cells express and secrete NGF.

The present disclosure also demonstrates that MSCs when introduced to asite, or near a site of brain injury and/or spinal cord injury, produceand secrete an array of factors including, but not limited to trophicfactors, cytokines and growth factors. The factors secreted by the MSCsserve to activate, among others, endogenous stem cells andsubepedymal/epedymal cells in the brain and/or spinal cord toproliferate and differentiate into parenchymal cells, including, but notlimited to neurons. Thus, the present invention includes a method ofusing MSCs to promote repair and plasticity of a CNS tissue in a mammalincluding, but not limited to brain and spinal cord that has undergonedisease, disorder or condition associated with a defect in the CNS.Preferably, the disease is MS.

The cells of the present invention can also be used to secrete anangiogenic factor including, but not limited to vascular growth factor,endothelial cell growth factor, and the like. MSCs can be used to induceangiogenesis within the tissue in which the MSCs are present. Thus, theinvention provides a method of promoting neovascularization within atissue using such cells. In accordance with this method, the cells areintroduced to the desired tissue under conditions sufficient for thecell to produce the angiogenic factor. The presence of the factor withinthe tissue promotes neovascularization within the tissue.

The mode of administration of the cells of the invention to the CNS ofthe mammal may vary depending on several factors including the type ofdisease being treated, the age of the mammal, whether the cells aredifferentiated or not, whether the cells have heterologous DNAintroduced therein, and the like. An example of administration of thecells into a brain tissue is provided herein in the experimentalExamples section. In that example, cells are introduced into the brainof a mammal by intracerebral or intravascular transplantation. Cells maybe introduced to the desired site by direct injection, or by any othermeans used in the art for the introduction of compounds into the CNS.

The cells can be administered into a host in a wide variety of ways.Preferred modes of administration are intravascular, intracerebral,parenteral, intraperitoneal, intravenous, epidural, intraspinal,intrastemal, intra-articular, intra-synovial, intrathecal,intra-arterial, intracardiac, or intramuscular. In some embodiments,MSCs are administered to the brain by direct transplantation asdescribed herein in the experimental Examples section. In otherembodiments, MSCs are administered to the central nervous system, i.e.,the spinal cord, by simple injection.

Transplantation of the cells of the present invention can beaccomplished using techniques well known in the art as well as thosedescribed herein or as developed in the future. The present inventioncomprises a method for transplanting, grafting, infusing, or otherwiseintroducing the cells into a mammal, preferably, a human. Exemplifiedherein are methods for transplanting the cells into brains of variousmammals, but the present invention is not limited to such anatomicalsites or to those mammals. Also, methods for bone transplants are wellknown in the art and are described in, for example, U.S. Pat. No.4,678,470, pancreas cell transplants are described in U.S. Pat. No.6,342,479, and U.S. Pat. No. 5,571,083, teaches methods fortransplanting cells to any anatomical location in the body.

In order to transplant the cells of the present invention into a mammal,for example a rat, the rat is anesthetized, preferably withapproximately 3.5% halothane, and anesthesia is maintained with 1.0%halothane in 70% N₂O and 30% O₂ or any cocktail well known in the art.The rat is then positioned on a stereotaxic instrument. A midlineincision is made in the scalp and a is hole drilled for the injection ofthe cells. Rats receive implants of the cells into the right striatumusing a glass capillary attached to a 10 μl Hamilton syringe. Each ratreceives a total of about 1×10⁵ cells. Following implantation, the skinwas sutured closed with either thread or staples. After recovery, therats are behaviorally tested and sacrificed for histological andimmunological analysis to determine the differentiation of both theimplanted cell and the endogenous cells of the CNS in vivo.

The cells of the present invention can be transplanted into a human.Preferably, the cells are from the patient for which the cells are beingtransplanted into (autologous transplantation). One preferable mode ofadministration is as follows. In the case where cells are not from thepatient (allogeneic transplantation), at a minimum, blood type orhaplotype compatibility should be determined between the donor cell andthe patient. Surgery is performed using a Brown-Roberts-Wells computedtomographic (CT) stereotaxic guide. The patient is given localanesthesia in the scalp area and intravenously administered midazolam.The patient undergoes CT scanning to establish the coordinates of theregion to receive the transplant. The injection cannula usually consistsof a 17-gauge stainless steel outer cannula with a 19-gauge innerstylet. This is inserted into the brain to the correct coordinates, thenremoved and replaced with a 19-gauge infusion cannula that has beenpreloaded with about 30 μl of tissue suspension. The cells are slowlyinfused at a rate of about 3 μl/min as the cannula is withdrawn.Multiple stereotactic needle passes are made throughout the area ofinterest, approximately 4 mm apart. The patient is examined by CT scanpostoperatively for hemorrhage or edema. Neurological evaluations areperformed at various post-operative intervals, as well as PET scans todetermine metabolic activity of the implanted cells.

Between about 10⁵ and about 10¹³ cells per 100 kg person areadministered to a human per infusion. In some embodiments, between about1.5×10⁶ and about 1.5×10¹² cells are infused per 100 kg person. In someembodiments, between about 1×10⁹ and about 5×10¹¹ cells are infused per100 kg person. In some embodiments, between about 4×10⁹ and about 2×10¹¹cells are infused per 100 kg person. In some embodiments, between about5×10⁸ cells and about 1×10¹⁰ cells are infused per 100 kg person.

In some embodiments, a single administration of cells is provided. Insome embodiments, multiple administrations are provided. In someembodiments, multiple administrations are provided over the course of3-7 consecutive days. In some embodiments, 3-7 administrations areprovided over the course of 3-7 consecutive days. In some embodiments, 5administrations are provided over the course of 5 consecutive days.

In some embodiments, a single administration of between about 10⁵ andabout 10¹³ cells per 100 kg person is provided. In some embodiments, asingle administration of between about 1.5×10⁸ and about 1.5×10¹² cellsper 100 kg person is provided. In some embodiments, a singleadministration of between about 1×10⁹ and about 5×10¹¹ cells per 100 kgperson is provided. In some embodiments, a single administration ofabout 5×10¹⁰ cells per 100 kg person is provided. In some embodiments, asingle administration of 1×10¹⁰ cells per 100 kg person is provided.

In some embodiments, multiple administrations of between about 10⁵ andabout 10¹³ cells per 100 kg person are provided. In some embodiments,multiple administrations of between about 1.5×10⁸ and about 1.5×10¹²cells per 100 kg person are provided. In some embodiments, multipleadministrations of between about 1×10⁹ and about 5×10¹¹ cells per 100 kgperson are provided over the course of 3-7 consecutive days. In someembodiments, multiple administrations of about 4×10⁹ cells per 100 kgperson are provided over the course of 3-7 consecutive days. In someembodiments, multiple administrations of about 2×10¹¹ cells per 100 kgperson are provided over the course of 3-7 consecutive days. In someembodiments, 5 administrations of about 3.5×10⁹ cells are provided overthe course of 5 consecutive days. In some embodiments, 5 administrationsof about 4×10⁹ cells are provided over the course of 5 consecutive days.In some embodiments, 5 administrations of about 1.3×10¹¹ cells areprovided over the course of 5 consecutive days. In some embodiments, 5administrations of about 2×10¹¹ cells are provided over the course of 5consecutive days.

In a one embodiment of the invention, the cells of the present inventionare administered to a mammal suffering from a disease, disorder orcondition involving the CNS, in order to augment or replace the diseasedand damaged cells of the CNS. MSCs are preferably administered to ahuman suffering from a disease, disorder or condition involving the CNS.The MSCs are further preferably administered to the brain or spinal cordof the human. In some instances, the cells are administered to theadjacent site of injury in the human brain. The precise site ofadministration of the cells depend on any number of factors, includingbut not limited to, the site of the lesion to be treated, the type ofdisease being treated, the age of the human and the severity of thedisease, and the like. Determination of the site of administration iswell within the skill of the artisan versed in the administration ofsuch cells. Based on the present disclosure, the cells can beadministered to the patient via intracarotid or intravenous routes.

In another embodiment, the therapeutic benefit of administering bonemarrow stromal cells to a mammal in need thereof is not the result of acell replacement therapy. That is, the administered bone marrow stromalcells provide a therapeutic benefit by inducing endogenous CNS cells toexpress and secrete a growth factor. In one aspect, the bone marrowstromal cells stimulate brain parenchymal cells to express and secrete afactor including, but not limited to NGF, BDNF, VEGF, and bFGF. By wayof example, the expression and secretion of NGF by the parenchymal cellsprovides an elevated level of NGF compared to the level of NGF presentin an otherwise identical CNS not treated with bone marrow stromalcells. In any event, the elevated level of NGF present in the CNSprovides a therapeutic benefit, including but not limited to stimulatingaxonal repair, prevent demyelination, reducing axonal loss, stimulatingoligodendrocyte growth, stimulated oligodendrocyte differentiation,enhancing survival of differentiated oligodendrocytes, and exhibitingimmunomodulatory effects.

There are several ways in which MSCs can be used in a mammal,preferably, a human, to treat diseases of the central nervous system.For example, the cells can be used as precursor cells that differentiatefollowing introduction into the CNS or as cells which have beendifferentiated into neural cells prior to introduction into the CNS. Ineither situation, the cells can be differentiated to express at leastone characteristic of a cell of the CNS including, but not limited toclass III β-tubulin, the M subunit of neurofiliments, tyrosinehydroxylase, glutamate receptor subunits of the GluR1-4 and GluR6classes, glial fibrillary acidic protein, myelin basic protein, brainfactor 1, NeuN, NF-M, NSE, nestin, and trkA.

The data presented herein establish that MSCs, when transplanted into amammal, express proteins characteristic of astrocytes (positive forglial fibrillary acidic protein, GFAP, a marker for early astrocytes)and neurons (positive for microtubule associate protein-2, MAP-2, amarker for neurons). It is anticipated that MSCs which are introducedinto the CNS can differentiate into other cell types including, but notlimited to oligodendrocytes, Schwann cells and parenchymal cells.

Further, the disclosure herein demonstrates that following introductionof MSCs into a mammal, the cells can secrete various factors. Suchfactors include, but are not limited to, growth factors, trophic factorsand cytokines. In some instances, the secreted factors can have atherapeutic effect in the mammal. The secreted factors can activate thecell from which the factor was secreted from. In addition, the secretedfactor can activate neighboring and/or distal endogenous cells toproliferate and/or differentiate. Preferably an MSC secretes a cytokineor growth factor such as human growth factor, fibroblast growth factor,nerve growth factor, insulin-like growth factors, hemopoietic stem cellgrowth factors, members of the fibroblast growth factor family, membersof the platelet-derived growth factor family, vascular and endothelialcell growth factors, members of the TGFβ family (including bonemorphogenic factor), or enzymes specific for congenital disorders.

MSCs can also secrete factors, trophic factors, and cytokines including,but not limited to, leukemia inhibitory factor (LIF), brain-derivedneurotrophic factor (BDNF), epidermal growth factor receptor (EGF),basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophicfactor (GDNF), granulocyte colony-stimulating factor (GCSF), hepatocytegrowth factor (HGF), IFN-γ, insulin-like growth factor binding protein(IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein(MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF),neurotrophic factors (NT3), tissue inhibitor of metalloproteinases(TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelialgrowth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor(uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stemcell factor (SCF), stromal cell-derived factor-1 (SDF-1), plateletderived growth factor-BB (PDGFBB), transforming growth factors betaTGFβ-1 and TGFβ-3.

The data presented herein establishes that the cells can successfullygraft to the CNS tissue. Further, the cells can migrate to differentregions within the brain including, but not limited to hippocampus,olfactory bulb and cortex. These cells may therefore replace cells inthe CNS which have been lost as a result of a genetic disease, trauma,or other injury. Further, these cells can activate endogenous cells toproliferate and/or differentiate.

In addition, prior to the introduction of the cells into the CNS, thecells may be genetically engineered to produce molecules such as trophicfactors, growth factors, cytokines, neurotrophins, and the likes, whichare beneficial to cells which are already present in the CNS. Forexample, MSCs can be cultured and genetically engineered cells prior totheir introduction into a recipient, and following the introduction ofthe engineered cell into the recipient, the cells are able to repair thedefected CNS tissue.

Based on these considerations, the types of diseases which are treatableusing the cells of the present are limitless. For example, amongneonates and children, the cells may be used for treatment of a numberof genetic diseases of the CNS, including, but not limited to, Tay-Sachsdisease and the related Sandhoff's disease, Hurler's syndrome andrelated mucopolysaccharidoses and Krabbe's disease. To varying extents,these diseases also produce lesions in the spinal cord and peripheralnerves. In addition, in neonates and children, treatment of head traumaduring birth or following birth is treatable by introducing the cellsinto the CNS of the individual. CNS tumor formation in children is alsotreatable using the methods of the present invention.

With respect to adult diseases of the CNS, the cells of the presentinvention are useful for treatment of Parkinson's disease, Alzheimer'sdisease, spinal cord injury, stroke, trauma, tumors, degenerativediseases of the spinal cord such as amyotropic lateral sclerosis,Huntington's disease, epilepsy and the like. Treatment of multiplesclerosis may also be possible.

Other neurodegenerative diseases include but are not limited to, AIDSdementia complex; demyelinating diseases, such as multiple sclerosis andacute transferase myelitis; extrapyramidal and cerebellar disorders,such as lesions of the ecorticospinal system; disorders of the basalganglia or cerebellar disorders; hyperkinetic movement disorders, suchas Huntington's Chorea and senile chorea; drug-induced movementdisorders, such as those induced by drugs that block CNS dopaminereceptors; hypokinetic movement disorders, such as Parkinson's disease;progressive supra-nucleopalsy; structural lesions of the cerebellum;spinocerebellar degenerations, such as spinal ataxia, Friedreich'sataxia, cerebellar cortical degenerations, multiple systemsdegenerations (Mencel, Dejerine Thomas, Shi-Drager, and Machado-Joseph),systermioc disorders, such as Rufsum's disease, abetalipoprotemia,ataxia, telangiectasia; and mitochondrial multi-system disorder;demyelinating core disorders, such as multiple sclerosis, acutetransverse myelitis; and disorders of the motor unit, such as neurogenicmuscular atrophies (anterior horn cell degeneration, such as amyotrophiclateral sclerosis, infantile spinal muscular atrophy and juvenile spinalmuscular atrophy); Alzheimer's disease; Down's Syndrome in middle age;Diffuse Lewy body disease; Senile Demetia of Lewy body type;Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakobdisease; Subacute sclerosing panencephalitis hallerrorden-Spatz disease;and Dementia pugilistica. See, e.g., Berkow et. al., (eds.) (1987), TheMerck Manual, (15^(th) edition), Merck and Co., Rahway, N.J., whichreference, and references cited therein, are entirely incorporatedherein by reference.

In some aspects of the invention, an individual suffering from adisease, disorder, or a condition that affects the CNS that ischaracterized by a genetic defect may be treated by supplementing,augmenting and/or replacing defective or deficient neurological cellswith cells that correctly express a normal neurological cell gene. Thecells which are to be introduced into the individual may be derived froma different donor (allogeneic) or they may be cells obtained from theindividual to be treated (autologous). In addition, the cells to beintroduced into the individual can by obtained from an entirelydifferent species (xenogeneic). The cells may also be geneticallymodified to correct the defect. But this is not the only instance wherethe cells can be genetically modified.

In another aspect of the invention, an individual suffering from adisease, disorder or a condition of the central nervous system can betreated as follows. Isolated MSCs are obtained and expanded in culture.The cells are then administered to the individual in need thereof. It isenvisioned that some of the isolated/expanded cells that areadministered to the individual develops into normal cells of the centralnervous system. Thus, repopulation of the central nervous system tissuewith an expanded population of MSCs serves to provide a population ofnormal central nervous system cells which facilitate correction of thedefect in the central nervous system tissue. In addition, the cells thatare introduced into the individual can secrete agents including, but notlimited to growth factors, trophic factors, cytokines and the like toactivate endogenous cells of the individual to proliferate anddifferentiate.

Based upon the disclosure herein, it is envisioned that the MSCs of thepresent invention can be administered to the individual in need thereofwithout the requirement of using immunosuppressive drug therapy. It isrecognized that cells from disparate individuals invariably results inthe risk of graft rejection. However, it was observed that MSCs did notinduce an immune response when the cells were administered to anallogeneic recipient. Further, it was observed that the presence of animmunosuppressive drug, for example cyclosporine A (CsA) duringtransplantation of MSCs to an allogeneic mammal, did not contributeanymore significant effects on neurological functional recovery comparedto when MSCs were administered to an otherwise identical mammal withoutreceiving an immunosuppressive drug. Therefore, as more fully discussedelsewhere herein, an aspect of the invention includes using allogeneicMSCs for transplantation.

The invention also includes methods of using MSCs of the presentinvention in conjunction with current mode, for example the use ofimmunosuppressive drug therapy, for the treatment of host rejection tothe donor tissue or graft versus host disease. An advantage of usingMSCs in conjunction with immunosuppressive drugs in transplantation isthat by using the methods of the present invention to ameliorate theseverity of the immune response following transplantation, the amount ofimmunosuppressive drug therapy used and/or the frequency ofadministration of immunosuppressive drug therapy can be reduced. Abenefit of reducing the use of immunosuppressive drug therapy is thealleviation of general immune suppression and unwanted side effectsassociated with immunosuppressive drug therapy.

In another aspect of the invention, the cells are pre-differentiatedinto, for example, neurons prior to administration of the cells into theindividual in need thereof. MSCs can be differentiated in vitro bytreating the cells with differentiation factors including, but are notlimited to antioxidants, epidermal growth factor (EGF), and brainderived neurotrophic factor (BDNF). It has been demonstrated thattreatment of the cells with these factors induced the cells to undergomorphologic changes consistent with neuronal differentiation, i.e., theextension of long cell processes terminating in growth cones andfilopodia. In addition, it was observed that these agents induced theexpression of neuronal specific proteins including, but are not limitedto nestin, neuron-specific enolase (NSE), neurofilament M (NF-M),neuron-specific nuclear protein (NeuN), and the nerve growth factorreceptor trkA.

Treating CNS Disorders

Treating a human patient having a disease, disorder, or a condition thataffects the CNS, encompasses among others, intracerebral grafting ofMSCs or MSC-differentiated cells to the CNS, including the region of theCNS having the injury or a region adjacent to the site of injury.MSC-differentiated cells include, for example, oligodendrocyteprecursors that have been differentiated by culturing MSCs in adifferentiation medium. The cells of the invention can be injected intoa number of sites, including the intraventricular region, the parenchyma(either as a blind injection or to a specific site by stereotaxicinjections), and the subarachnoid or subpial spaces. Specific sites ofinjection can be portions of the cortical gray matter, white matter,basal ganglia, and spinal cord. Without wishing to be bound to anyparticular theory, any mammal affected by a CNS disorder, as describedelsewhere herein, can be so treated by one or more of the methodologiesdescribed herein.

Conventional techniques for grafting are described, for example, inBjorklund and Stenevi (1985, Neural Grafting in the Mammalian CNS, eds.Elsevier, pp 169-178), the contents of which are incorporated byreference. Procedures include intraparenchymal transplantation, achievedby injecting the cells of the invention into the host brain tissue.However, transplantation of the cells of the invention can be effectedin a number of CNS regions.

According to the present invention, administration of cells intoselected regions of a patient's brain may be made by drilling a hole andpiercing the dura to permit the needle of a microsyringe to be inserted.Alternatively, the cells can be injected intrathecally into a spinalcord region. The cell preparation of the invention permits grafting ofthe cells to any predetermined site in the brain or spinal cord. It alsois possible to effect multiple grafting concurrently, at several sites,using the same cell suspension, as well as mixtures of cells.

The data disclosed herein demonstrate the effects of administering thecells of the present invention to a mammal that has undergone a disease,disorder, or a condition that affects the CNS, otherwise a CNS injury,for example stroke using intraarterial (IA) or intravenous (IV) deliverysystems. The effects of MSCs injected via IA or IV in an injured mammalwas assessed by analyzing neurological function, neurogenesis, andangiogenesis in mammals that were subjected to ischemic conditions.Quantitative analysis using immunohistochemistery techniques indicatedthat angiogenesis was significantly enhanced by the administration ofthe cells of the present invention. The data disclosed hereindemonstrated that no significant differences were observed with respectto neurological function, neurogenesis, and angiogenesis in mammals thatreceived either IA or IV administration of the cells. Based on thepresent disclosure, MSCs delivered to the ischemic brain through bothintracarotid and intravenous routes provide therapeutic benefits to amammal that has undergone stroke. However, the invention should in noway be construed to be limited to any one method of administering MSCs.Rather, any method of administration of the cells should be construed tobe included in the present invention. Further, the invention should inno way be limited to stroke, rather, any disease, disorder or conditionof the CNS can be treated using compositions and methods of the presentinvention.

Treatment of a patient, according to the invention, can take advantageof the migratory ability of MSCs, and using them to provide a peptide,protein or other substance to a region of the CNS affected by adysfunction or deficiency relating to that substance. As such, the cellsof the invention may contain exogenous DNA encoding a product that ismissing in an individual suffering from a CNS disorder. For example, theDNA can code for a transmitter, such as acetylcholine or GABA, or areceptor for such a transmitter. If an individual is suffering from aglutamate-induced injury, it may be desirable to introduce into thepatient a gene coding for a glutamate transporting protein, which canreduce glutamate-induced cytotoxicity.

In a further approach, DNA that encodes a growth factor or a cytokinecan be transfected to MSCs, which then are administered to a patientsuffering from a CNS disorder, the etiology or elaboration of which isassociated with a deficit or dysfunction in the gene expression product.To this end, the invention includes, for example, the use of a genethat, upon expression, produces factors including, but are not limitedto NGF, brain-derived neurotrophic factor (BDNF), glial cellline-derived neurotrophic factor (GDNF), insulin-like growth factor(IGF-1) and ciliary neurotrophic factor (CNTF). In addition, theselected gene can encode leukemia inhibitory factor (LIF) or any otherof the other cytokines, disclosed, for example, by Reichardt et al.(1997, Molecular and Cellular Approaches to Neural Development, OxfordUniversity Press: 220-263), supra, that promotes cell survival ordifferentiation.

A therapeutic procedure according to the present invention can beeffected by injecting cells, preferably stereotaxically, into the cortexor the basal ganglia. Thereafter, the diffusion and uptake of a ligandsecreted by an MSC is beneficial in alleviating the symptoms of adisorder where the subject's neural cells are defective in theproduction of such a gene product. Thus, an MSC genetically modified tosecrete a neurotrophic factor, such as nerve growth factor (NGF), isused to prevent degeneration of cholinergic neurons that might otherwisedie without treatment. Alternatively, MSCs to be grafted into a subjectwith a disorder characterized by a loss of dopamine neurons, such asParkinson's disease, can be modified to contain exogenous DNA encodingL-DOPA, the precursor to dopamine.

According to the present invention, other CNS disorders likewise can betreated, including Alzheimer's disease, ganglioside storage diseases,CNS damage due to stroke, and damage in the spinal cord. For example,Alzheimer's disease is characterized by degeneration of the cholinergicneurons of the basal forebrain. The neurotransmitter for these neuronsis acetylcholine, which is necessary for their survival. Engraftment ofan MSC containing an exogenous gene encoding for a factor that wouldpromote survival of these neurons, can be accomplished by the method ofthe invention described herein.

The use of MSCs for the treatment of a disease, disorder, or a conditionthat affects the CNS provides an additional advantage in that the MSCscan be introduced into a recipient without the requirement of animmunosuppressive agent. Successful transplantation of a cell isbelieved to require the permanent engraftment of the donor cell withoutinducing a graft rejection immune response generated by the recipient.Typically, in order to prevent a host rejection response, nonspecificimmunosuppressive agents such as cyclosporine, methotrexate, steroidsand FK506 are used. These agents are administered on a daily basis andif administration is stopped, graft rejection usually results. However,an undesirable consequence in using nonspecific immunosuppressive agentsis that they function by suppressing all aspects of the immune response(general immune suppression), thereby greatly increasing a recipient'ssusceptibility to infection and other diseases.

The present invention provides a method of treating a disease, disorder,or a condition that affects the CNS by introducing MSCs into therecipient without the requirement of immunosuppressive agents. Thepresent invention relates to the discovery that administration of anallogeneic or a xenogeneic MSC, or otherwise an MSC that is geneticallydisparate from the recipient, into a recipient provides a benefit to therecipient. The present disclosure demonstrates that administration ofsuch an MSC into a mammal that was subjected to MCAo (to induce strokeconditions) did not exhibit host rejection to the MSC. The disclosurepresented herein demonstrates that administration of MSCs into thediseased mammal exhibit significant neurological recovery as measured byAdhesive-Removal and mNSS tests without the observation of the MSCsinducing a cytotoxic T lymphocyte response. Further, there was nosignificant difference in the neurological recovery between groups thatreceived transplantation of MSCs in the presence or absence of animmunosuppressive agent such as cyclosporine. Thus, the presentinvention provides a method of administering MSCs to a recipient havinga disease, disorder, or a condition that affects the CNS withoutinducing an immune response by the recipient against the MSCs.Therefore, the present invention provides a method of using MSCs totreat a disease, disorder or condition without the requirement of usingimmunosuppressive agents when administering MSCs to a recipient. Thereis, therefore, a reduced susceptibility for the recipient of thetransplanted MSCs to incur infection and other diseases, includingcancer relating conditions that is associated with immunosuppressiontherapy.

Genetic Modification

The cells of the present invention can also be used to express a foreignprotein or molecule for a therapeutic purpose or for a method oftracking their integration and differentiation in a patient's tissue.Thus, the invention encompasses expression vectors and methods for theintroduction of exogenous DNA into the cells with concomitant expressionof the exogenous DNA in the cells such as those described, for example,in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York).

The isolated nucleic acid can encode a molecule used to track themigration, integration, and survival of the cells once they are placedin the patient, or they can be used to express a protein that ismutated, deficient, or otherwise dysfunctional in the patient. Proteinsfor tracking can include, but are not limited to green fluorescentprotein (GFP), any of the other fluorescent proteins (e.g., enhancedgreen, cyan, yellow, blue and red fluorescent proteins; Clontech, PaloAlto, Calif.), or other tag proteins (e.g., LacZ, FLAF-tag, Myc, His₆,and the like) disclosed elsewhere herein. Alternatively, the isolatednucleic acid introduced into the cells can include, but are not limitedto CFTR, hexosaminidase, and other gene-therapy strategies well known inthe art or to be developed in the future.

Tracking the migration, differentiation and integration of the cells ofthe present invention is not limited to using detectable moleculesexpressed from a vector or virus. The migration, integration, anddifferentiation of a cell can be determined using a series of probesthat would allow localization of transplanted MSCs. Such probes includethose for human-specific Alu, which is an abundant transposable elementpresent in about 1 in every 5000 base pairs, thus enabling the skilledartisan to track the progress of the transplanted cell. Trackingtransplanted cell may further be accomplished by using antibodies ornucleic acid probes for cell-specific markers detailed elsewhere herein,such as, but mot limited to, NeuN, MAP2, neurofilament proteins, and thelike.

The invention also includes an MSC which, when an isolated nucleic acidis introduced therein, and the protein encoded by the desired nucleicacid is expressed therefrom, where it was not previously present orexpressed in the cell or where it is now expressed at a level or undercircumstances different than that before the transgene was introduced, abenefit is obtained. Such a benefit may include the fact that there hasbeen provided a system wherein the expression of the desired nucleicacid can be studied in vitro in the laboratory or in a mammal in whichthe cell resides, a system wherein cells comprising the introducednucleic acid can be used as research, diagnostic and therapeutic tools,and a system wherein mammal models are generated which are useful forthe development of new diagnostic and therapeutic tools for selecteddisease states in a mammal.

A cell expressing a desired isolated nucleic acid can be used to providethe product of the isolated nucleic acid to another cell, tissue, orwhole mammal where a higher level of the gene product can be useful totreat or alleviate a disease, disorder or condition associated withabnormal expression, and/or activity. Therefore, the invention includesan MSC expressing a desired isolated nucleic acid where increasingexpression, protein level, and/or activity of the desired protein can beuseful to treat or alleviate a disease, disorder or condition involvingthe CNS.

The MSC can be genetically engineered to express a growth factor, forexample NGF, prior to the administration of the engineered MSC into therecipient. The engineered MSC expresses and secretes NGF at a largeramount compared with an MSC that has not been genetically modified toexpress such a factor. A benefit of using a genetically modified MSC inthe treatment of a disease, disorder, or a condition that affects theCNS is to increase the therapeutic effects of having MSCs present in therecipient. The increased therapeutic effect is attributed to theincrease secretion of NGF from the engineered MSC. With the increasedsecretion of NGF from the engineered MSC, a larger amount of NGF ispresent for neighboring cells or distal cells to benefit from the NGF.In addition, the increase amount of NGF present in the recipient allowsa decrease in the time frame from which a patient can be treated.

Methods of Affecting/Modulating Cell Survival

The skilled artisan, when armed with the disclosure herein, can readilyappreciate that the present invention encompasses novel methods andcompositions for modulating/affecting cell survival, for example,increasing cellular survival through increased Akt and Erk1 at both theprotein level and the RNA level. The present invention is based on thediscovery that when MSCs were co-cultured with astrocytes that have beensubjected to ischemic conditions, it has been observed that the presenceof MSCs with the post-ischemic astrocyte in culture reduced the amountof cell death of the astrocytes. The present disclosure demonstratesthat when astrocytes were incubated in ischemic conditions such asincubation of the astrocytes in an anaerobic chamber, and thenco-cultured with MSCs, there was a significant reduction in the celldeath and apoptotic phenotypes exhibited by the astrocytes compared withpost-ischemic astrocytes cultured in the absence of MSCs.

The data herein demonstrates that co-culturing astrocytes with MSCsupregulated the phosphorylation of Erk1 and Akt in astrocytes. While notwishing to be bound to any particular theory, it is believed that MSCscontribute to the survival of neighboring astrocytes by activatingcellular proliferation and survival signaling pathwayspost-translationally. It was demonstrated that astrocytes that weretreated with MEK inhibitor (U0126), which inhibits Erk1 phoshporylation,or PI3K inhibitor (LY29004), which inhibits Akt phosphorylation,underwent significant apoptosis and cell death similar to thepost-ischemic control group. As such, the inhibition of molecularpathways leading to the activation of Erk1 and/or Akt inhibits theability of a cell to survive conditions that cause cell death and/orapoptosis that otherwise activation of such pathways would overcome thecell death and/or apoptosis conditions. Based on the present disclosure,the duration for which Erk1 and/or Akt are activated increases theability of a cell that has been subjected to cell death/apoptoticconditions to survival from such conditions. In addition, the intensityfor which Erk1 and/or Akt is activated in a cell increases the survivalpotential of a cell from cell death/apoptotic conditions. As such, thepresent invention comprises a method of using MSCs to activate survivalsignals, such as activation of Erk1 and/or Akt in a cell in order toconfer protection to the cell from cell death/apoptotic conditions.

In addition to the ability of MSCs to activate cellular pathways inneighboring cells, the present disclosure also demonstrates that MSCscan induce neighboring post-ischemic astrocytes to increase thetranscription of various growth factors including, but are not limitedto bFGF, BDNF, and VEGF. Based upon the present disclosure, one skilledin the art would appreciate that MSCs can enhance the recovery ofpost-ischemia astrocytes by stimulating the activation of MEK/Akt andPI3K/Erk pathways in astrocytes, and increasing growth factor productionby astrocytes.

Axonal and Myelination Remodeling

Axonal loss and demyelination are frequently observed to be associatedwith a disease, disorder, or a condition that affects the CNS. Axonalloss and demeylination is believed to contribute to neurologicalfunctional impairment in CNS conditions, for example, in ischemiccerebrovascular diseases and inflammatory demyelinating diseases, suchas MS.

The disclosure presented herein demonstrates that administration of MSCsto a diseased mammal having a condition including, but not limited to anischemic condition, an axonal degeneration, or demyelination, improvesneurological functional recovery in the diseased mammal. The presentdisclosure also demonstrates that the administered cells play a role inthe formation and/or maintenance of axonal fibers in an injured orotherwise diseased brain. In some instances, the administered cellsprevent axonal fiber loss in the brain of the diseased mammal. Mammalsthat were subjected to ischemic conditions or conditions of inflammatorydemyelination and subsequently treated with the administration of MSCsdemonstrated significant reduced areas of demyelination and reducedareas of axon loss compared with an otherwise identical mammal nottreated with MSCs. While not wishing to be bound to any particulartheory, the improved neurological functions exhibited by the diseasedmammal following treatment with MSCs is attributed to the reduceddemyelination and axon loss.

In some instances, the therapeutic effects of administering MSCs to themammal suffering from a neurodegenerative disease, such as MS, is notthe result of cell replacement therapy. That is, the disclosurepresented herein demonstrates that a therapeutic effect from theadministered MSCs can be attributed to the fact that the MSCs stimulateendogenous brain parenchymal cells to express NGF. The expression of NGFby endogenous brain parenchymal cells stimulates axonal repair in bothacute and chronic diseases. Thus, in some instances, a therapeuticoutcome from transplantation of MSCs to a mammal in need thereof doesnot require differentiation of MSCs into cells of the CNS, such asneurons, to replace the damaged or injured cells.

Based on the present disclosure, one skilled in the art would appreciatethat the loss of axonal fibers and demyelination of the axons contributeto neurological impairment. Axons play a major role in the neurologicalfunctions of a mammal. Axons are insulated by a myelin sheath, whichgreatly increases the rate at which an axon can transport a signal. Anyloss in axonal fibers or conditions of demeylination retards neuronsfrom properly functioning, for example, significant impairment insensory, motor and other types of functioning when nerve signals reachtheir targets either too slowly, asynchronously (when some axons in anerve conduct faster than others), intermittently (when conduction isimpaired only at high frequencies), or not at all. As such, the presentinvention provides compositions and methods for treating a neurologicalimpairment by preventing degradation of axonal fibers and preventingdemyelination. In addition, the present invention encompassescompositions and methods for using MSCs to remodel axonal fibers andmyelination.

The above discussion provides a factual basis for the use of bone marrowstromal cell transplantation for the treatment of neural injury andneurodegeneration. The methods used with and the utility of the presentinvention can be shown by the following non-limiting examples andaccompanying figures.

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and in Watson et al., Recombinant DNA, Scientific AmericanBooks, New York and in Birren et al (eds) Genome Analysis: A LaboratoryManual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York(1998) and methodology as set forth in U.S. Pat. Nos. 4,66,828;4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein byreference. Polymerase chain reaction (PCR) was carried out generally asin PCR Protocols: A Guide To Methods And Applications, Academic Press,San Diego, Calif. (1990). In-situ (in-cell) PCR in combination with FlowCytometry can be used for detection of cells containing specific DNA andmRNA sequences (Testoni et al, 1996, Blood 87:3822.).

Standard methods in immunology known in the art and not specificallydescribed are generally followed as in Stites et al. (eds), Basic andClinical Immunology (8th Edition), Appleton & Lange, Norwalk, Conn.(1994) and Mishell and Shiigi (eds), Selected Methods in CellularImmunology, W.H. Freeman and Co., New York (1980).

EXAMPLES Example 1 Treatment of Stroke (Rat) with IntracerebralTransplantation of MSC Description of Intracerebral Transplantation ofBone Marrow Derived MSCs After Cerebral Ischemia in the Rat

Adult male Wistar rats were used in this study (n=28). Rats weresubjected to middle cerebral artery occlusion (MCAo) for two hours usingthe intraluminal occlusion model. Following MCAo, the control group(rats subjected to MCAo without receiving transplantation of MSCs(n=8)), was compared with the experimental groups, which includedinjection into the ischemic boundary zone (IBZ) at 24 hours after MCAo:phosphate buffered saline (n=4); non NGF cultured bone marrow MSCs(n=8); and NGF cultured MSCs (n=8). Approximately 4×10⁴ cells in 10 μltotal fluid volume were injected into the rat following MCAo. The ratswere sacrificed 14 days after MCAo.

Behavioral Outcome Measurements

Behavioral data from the battery of functional tests (rotarod,adhesive-removal and Neurological Severity Score tests (NSS))demonstrated that motor and somatosensory functions were impaired by theischemic insult by way of subjecting the rats to MCAo. It was observedthat no significant differences of the rotarod, adhesive-removal and NSStests were detected among the groups prior to surgery and beforetransplantation. Significant recovery of somatosensory behavior (p<0.05)and NSS (p<0.05) were detected in mammals transplanted with MSCsfollowing MCAo compared with mammals not receiving transplantation ofMSCs following MCAo mammal (FIGS. 1A, 1C). Mammals that receivedtransplantation of MSCs that were cultured with NGF displayedsignificant recovery in motor (p<0.05), somatosensory (p<0.05) and NSS(p<0.05) behavioral tests at two weeks post-transplantation with NGF,compared with transplantation of MSCs alone. FIGS. 1A, 1B, 1C show datafrom the adhesive-removal test, the rotarod-motor test and the NSS,respectively. These data clearly demonstrate that treatment of strokewith intracranial transplantation of MSCs provides significanttherapeutic benefit and that MSCs when cultured in NGF provides superiortherapeutic benefit compared with MSCs cultured without NGF, asindicated in the motor test data (FIG. 1B).

Example 2 Treatment of Stroke (Mouse) with Intracerebral Transplantationof MSC Intrastriatal Transplantation of MSCs into Mice After Stroke:Embolic MCAo and Transplantation

Experimental adult mice (C57BL/6, weighing about 27-35 g) were subjectedto MCAo and following MCAo, the mice received transplantation of MSCs(n=5). Control mice were subjected to MCAo alone (n=8). Experimentalgroups received either injection of PBS into the ischemic striatum(n=5); or transplantation of MSCs into the normal striatum (n=5). MCAowas induced using an embolic model developed in our laboratory (Zhang etal., 1997). Briefly, using a facemask, mice were anesthetized with 3.5%halothane and anesthesia was maintained with 1.0% halothane in 70% N₂Oand 30% O₂. A single intact fibrin-rich in 24 hour old homologous clot(8 mm×0.000625 mm², 0.18:1) was placed at the origin of the MCAo via amodified PE-50 catheter. Surgical and physiological monitoringprocedures were identical to those previously published (Zhang et al.,1997). Four days after MCAo (n=18), the mice were mounted on astereotaxic frame (Stoelting Co. Wood Dale, Ill.). Using aseptictechnique, a burr hole (1 mm) was made on the right side of the skull toexpose the dura overlying the right cortex. Semisuspended MSCs (1×10⁵ in3:1 PBS) were slowly injected over a 10-minute period into the rightstriatum (AP=0 mm, ML=2.0 mm, and DV=3.5 mm from the bregma). Withoutwishing to be bound to any particular theory, this position approximatesthe ischemic boundary zone in the striatum. The needle was retained inthe striatum for an additional 5 minutes interval to avoid donor reflux.Mice were sacrificed at 28 days after stroke.

Behavioral Testing

Each mouse was subjected to a series of behavioral tests (rotarod-motortest, Neurological Severity Score) to evaluate various aspects ofneurological function by an investigator who was blinded to theexperimental groups. Measurements were performed prior to stroke and at28 days after stroke.

Results

BrdU reactive MSCs survived and migrated a distance of approximately 2.2mm from the grafting areas toward the ischemic areas. BrdU reactivecells expressed neuronal (˜1% NeuN) and astrocytic proteins (˜8% glialfibrillary acidic protein, GFAP). Functional recovery from a rotarodtest (p<0.05) and modified Neurological Severity Score tests (NSS,including motor, sensory and reflex, p<0.05) were significantly improvedin the mice receiving MSCs following MCAo treatment compared with micenot receiving MSCs following MCAo treatment (FIG. 2). FIG. 2 shows thatmice treated with transplanted MSCs exhibited a significant improvementin the duration on the rotarod (FIG. 2) and an improved neurologicalfunction (FIG. 2) compared to vehicle treated mammals. The findingssuggest that the intrastriatal transplanted MSCs survive in the ischemicbrain and improve functional recovery of adult mice.

Example 3 Treatment of Stroke (Mouse) with Intravascular Administrationof MSC Description of Experiments

Experiments were performed on adult male Wistar rats (n=30) weighingabout 270 to 290 g. In all surgical procedures, anesthesia was inducedin rats with 3.5% halothane, and maintained with 1.0% halothane in 70%N₂O and 30% O₂ using a face mask. The rectal temperature was controlledat 37° C. with a feedback regulated water heating system. Transient MCAowas induced using a method of intraluminal vascular occlusion, asdescribed above. Two hours after MCAo, reperfusion was performed bywithdrawal of the suture until the tip cleared the internal carotidartery.

Intracarotid Administration of MSCs

Intra-carotid transplantation of MSCs was carried out at 24 hours afterMCAo (n=23). A modified PE-50 catheter was advanced from the same siteof this external carotid artery into the lumen of the internal carotidartery until it rested 2 mm proximal to the origin of the MCA (FIG. 1).Approximately 2×10⁶ MSCs in 200 μl PBS (n=6) or control fluid (200 μlPBS, n=8) were injected over a 10-minute period into each experimentalrat. Immunosuppressants were not used in any mammal. All rats weresacrificed at 14 days after MCAo.

Intravenous Administration of MSCs

For intravenous administration of MSCs, a femoral vein was cannulatedand either 1.5×10⁶ MSCs or 3×10⁶ MSCs were injected.

Behavioral Tests and Immunohistochemistry

Each rat was subjected to a series of behavioral tests (NSS and adhesiveremoval test) to evaluate neurological function before MCAo, and at 1,4, 7 and 14 days after MCAo. Single and double immunohistochemistry wereemployed to identify cell specific proteins of BrdU reactive MSCs.

Results

For intra-arterial administration, BrdU reactive cells (˜21% of 2×10⁶transplanted MSCs) distributed throughout the territory of the MCAo by14 days after ischemia. Some BrdU reactive cells expressed proteinscharacteristic of astrocytes (glial fibrillary acidic protein, GFAP) andneurons (microtubule associated protein-2, MAP-2). Rats with MSCintra-arterial transplantation exhibited significant improvement on theadhesive-removal test (p<0.05) (FIG. 3) and the modified NeurologicalSeverity Scores (p<0.05) (FIG. 3) at 14 days, compared with controls.The data for intravenous administration of MSCs were very similar, inthat significant functional improvement was present with rats treatedwith MSCs compared to placebo treated rats. FIG. 4 shows functional datafrom rats receiving administration of MSCs intravenously compared tocontrol-ischemia rats not receiving MSCs. A significant improvement isnoted in the speed in which the rats removed the sticky tabs from theirpaws at seven and 14 days after stroke, compared to control mammals(FIG. 4). The overall neurological function of rats receiving MSCsadministered intraarterially was significantly improved compared tocontrol-ischemia rats at 14 days after stroke. The findings suggest thatMSCs injected intra-arterially are localized and directed to theterritory of MCAo and these cells foster functional improvement aftercerebral ischemia. In addition, intravenous administration of MSCs alsoprovides a significant improvement in functional outcome. Thus, the datapresented demonstrated that vascular administration is a feasible andeffective route of administration of therapeutically beneficial MSCs.

Example 4 Treatment of Traumatic Brain Injury (Rat) with IntracerebralTransplantation of MSC Description

Experiments were performed on 66 male Wistar rats weighing about 250-350grams. A controlled cortical impact device was used to induce injury tothe rats (Dixon et al. 1991 J. Neuroscience Methods 39:253-262). Injurywas induced by impacting the left cortex with a pneumatic pistoncontaining a 6 mm diameter moving at a rate of 4 mm/second and producing2.5 mm compression. BrdU labeled MSCs were harvested from donor mammalsand implanted into the ipsilateral hemisphere, as in the strokeexperiments. MSCs were transplanted into brain 24 hours after injury.Rats receiving transplantation of MSCs were sacrificed at 4 days (n=4),1 week (n=15), 2 weeks (n=4) and 4 weeks (n=4) after transplantation.Control mammals were divided into 3 groups: 1) rats subjected to injurywithout transplantation and sacrificed at 8 days (n=4) and 29 days (n=4)after injury; 2) mammals injected with PBS one day after injury andsacrificed at 4 days (n=4), 7 days (n=4), 14 days (n=4) and 28 daysafter PBS injection; 3) sham control rats with craniotomy but no injuryor transplantation were sacrificed 8 days (n=4) and 29 days (n=4) aftercraniotomy.

Outcome Measures (Behavior, Histology)

An accelerating rotarod test was employed to measure motor function.Measurements were performed at 2, 5, 15, and 29 days after injury. Aftersacrifice, brain sections were stained with hematoxylin and eosin anddouble-labeled immunohistochemistry was performed to identify MSC celltype.

Results

Histological examination revealed that after transplantation MSCssurvived, proliferated and migrated towards the injury site. BrdUlabeled MSCs expressed markers for astrocytes and neurons. Ratstransplanted with MSCs exhibited a significant improvement in motorfunction compared with control mammals which did not receivedtransplantation of MSCs. The data indicate that intracerebraltransplantation of MSCs significantly improves neurological functionafter traumatic brain injury. In a complementary set of experiments,treated rats were also subjected to traumatic brain injury and receivedtransplantation of MSCs; however, in this experiment MSCs were deliveredto the brain by means of intraarterial (intracarotid artery)administration. The data were observed to be similar to intracranialtransplantation. MSCs migrated readily into the injured region of brainand these cells expressed protein markers of brain cells (astrocytes,neurons). Thus, the present disclosure indicate that traumatic braininjury can be treated with MSC administered intracerebrally or via avascular route.

Example 5 Treatment of Parkinson's (Mouse) with IntracranialTransplantation of MSCs Description of MPTP Method and Results

Adult male C57BL/6 mice, 8 week-old, weighing about 20-35 g, wereemployed in this study. In order to obtain severe and long lastinglesions, mice were treated with intraperitoneal injections of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride (30mg/kg, Sigma) in saline once a day for seven consecutive days (210 mg/kgtotal dose). Mice were transplanted with BrdU labeled MSCs (3×10⁵/3 μl)directly into the right striatum, stereotaxically.

Behavioral Tests

Mice subjected to each MPTP injection, presented and retained behavioralabnormalities (akinesia, postural instability, tremor and rigidity) forseveral hours, as reported in literature (Heikkila et al., 1989).

Drug-free evaluation of Parkinson's disease (PD) using rotarod test wasdescribed by Rozas et al. (1997, 1998). MPTP-PD mice with or without MSCtransplantation were tested on a rotarod at an increasing speed (16rev/minute and 20 rev/minute) after the last MPTP injections (fivetrials per day to obtain stable values) without any additional enhanceddrug injection. A trial was terminated when the mice fell from therotarod. Significant improvement in motor function (p<0.05) was observedat 35 days after MPTP injection in Parkinson's disease mice treated withMSC transplantation compared with control MPTP-injected mice alone. FIG.5 shows rotarod data from mice subjected to MPTP neurotoxicity. Twoexperiments were performed; the mice were placed on the rotarod rotatingat 16 rpm or at 20 rpm. The data demonstrated that mice treated withMSCs showed a significant increase in duration on the rotarod at bothangular velocities compared to MPTP mice given PBS intracerebrally. Micetreated with MSCs cultured with NGF appeared to have incremental benefitcompared to MSC treatment, although the differences were not observablysignificant.

Morphological Changes

Viable BrdU immunoreactive cells were identified in the injected areaand migrated to variable distances into the host striatum (FIG. 1B) at35 days. Double staining shows that scattered BrdU reactive cells (FIG.1C) express tyrosine hydroxyls (a dopamine marker) immunoreactivity(FIG. 1D) within the grafts.

Conclusions

These data demonstrate that intracerebral transplantation of MSCsreduces Parkinson's disease symptoms in mice.

Example 6 Treatment of Spinal Cork Injury (Rat) with IntralesionalTransplantation of MSCs Description of Spinal Cord Injury (SPI)

Impact injury was induced using the weight-drop 10 g from a height of 25mm, ‘NYU impact’ model) to produce a spinal cord injury of moderateseverity. Adult male Wistar rats (300±5 g) were anesthetized withpentobarbital (50 mg/kg, intraperitoneally), and a laminectomy wasperformed at the T9 level.

Transplantation and Behavioral Testing

MSCs 2.5×10⁵/4:1 were injected into the epicenter of injury at 7 daysafter SPI. The Basso-Beatie-Bresnahan (BBB) Locomotor Rating scores wereobtained before and after transplantation (Basso et al., 1995). FIG. 7shows data from the BBB test from mammals subjected to spinal cordinjury, and receiving MSC transplantation or simply given the samevolume of vehicle. All rats had a score of 21 (normal score) beforespinal cord injury and a score of zero at 6 hours after contusion. Inthe rats subjected to contusion with PBS injection, scores improved from6.7 (1 week) to 11.5 (5 weeks). The control group had an earlyimprovement in neurologic function, which plateaued by the third week.The rats subjected to contusion with MSC transplantation had asignificantly improved score of 7.0 (1 week) and 15.3 (5 weeks). The MSCtreated group exhibited a steady recovery that had not plateaued by thefifth weeks, which was the end point of the experiment. The MSC treatedrats had significant improvement on BBB scores with the p-value, 0.01for overall and each individual time point for treatment effect. Infunctional terms, the contused rats in the MSC treated group could walkwith consistent weight supported plantar steps with forelimb andhindlimb coordination. In contrast, the contused rats in the PBS controlgroup exhibited obvious motor function deficits.

Histological Analysis

Cells derived from MSCs, identified by BrdU immunoreactivity, survivedand were distributed throughout the damaged tissue (T9, FIG. 1A) from 1week to 4 weeks after MSC transplantation. BrdU reactive cells migrated5 mm both caudal and rostral from the epicenter of transplanted cells(FIG. 1B). FIG. 2A shows that the antibody against Rip did not reactwith damaged oligodendrocytes in contused rats with non treated PBSinjection. In contrast, after spinal cord injury and receiving MSCtransplantation (FIG. 2B), intense Rip immunoreactivity clearlydemarcated myelinated small and large diameter fibers. Doubleimmunostaining (FIGS. 2C-D) demonstrates that scattered BrdU reactivecells express the neuronal marker, NeuN.

Conclusions

Treatment of moderate to severe spinal cord injury in a mammal byadministering MSCs into the site of injury provides significantimprovement of motor function. The MSCs express protein markers ofneurons and oligodendrocytes, indicating that these cells when placedwithin the spinal cord acquire characteristics of parenchymal cells.

Example 6 Neurosphere (NMCsphere)—a New Composite for the Treatment ofCNS Injury and Disease Description of Neurosphere Experiment

Aggregates, composed of neural stem cells from fetal neurosphere,mesenchymal stem cells from adult bone marrow and cerebro-spinal fluidfrom adult Wistar rats (called NMCspheres) were used in the followingexperiments. Fetal brain cells were pre-labeled with1,1′-dioctadecy-6,6′-di(4-su1fophey1)-3,3,3′,3′-tetramethylindocarbocyanine(Dil) and bone marrow mesenchymal cells from adult rats were pre-labeledwith 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) and/orbromodeoxyuridine (BrdU). Using laser scanning confocal microscopy(three-dimensional) and immunohistochemical analysis on paraffin andfrozen sections, it was identified that:

1. Cell-cell interaction: Within the NMCsphere, cells derived from bonemarrow mesenchymal stem cells, rapidly form a scaffold (1 day) and anetwork (9 days, FIG. 8) overtime, in vitro. FIG. 8 depicts thecomposite MSC neurosphere nine days after cell-neurosphere integration.The MSC, identified by DO and BrdU form an axonal-dendritic like network(yellow-green).2. Cell-cell interaction: Within the NMCsphere, cells derived fromneural stem cells have a longer life span than within neurosphere alone.The NMCspheres express proteins, i.e., nestin that is normally found inimmature neural cells; glial fibrillary acidic protein (GFAP) that is aspecific marker for differentiated astrocytes; myelin basic protein(MBP) that is a marker of oligodendrocytes; and neuron-specific classIII β-tubulin (TuJ1) that is a marker for immature neurons andmicrotubule associated protein 2 (MAP-2) that is a marker for neuronalcell bodies and dendrites.3. NMCsphere-microenvironment: The size and structure of the NMCspheresare influenced by the microenvironment of the medium, i.e., they growbetter in the IMDM with stem cell factor than with standard DMEM.4. Secretion of NMCspheres: Adding the supernatant from the culturedNMCsphere into the medium DMEM and IMDM for neurospheres and MSCs,respectively, stimulated the growth of both neurospheres and MSCs.Obvious cell-cell connection and proliferation was induced with thissupernatant. This suggests the NMCspheres secrete supporting substancesfor stem cells. These substances can be used to enhance neurogenesis.5. Cerebro-spinal fluid (CSF) provides an optimal microenvironment toform NMCspheres that is superior to conventional medium.

Example 7 Treatment of Stroke and Brain Trauma with NMCsphere

Protocol for MSC & neurosphere transplantation in rats after MCAo andtraumatic brain injury (TBI).

MCAo

BrdU prelabeled MSCs and neurospheres were mixed and cultured in flasksfor 7 days. At 24 hours after MCAo, rats were anesthetized withhalothane and the composite NMSsphere was injected into the brain ofMCAo rats (n=4). The mammals were mounted on a stereotaxic apparatus(Model 51603, Stoelting Co., Wood Dale, Ill.). Twenty spheres (diameterless than 0.2 mm) in 5 ml PBS were injected vertically by a Hamiltonsyringe into the right striatum at the coordinates LM=2.5 mm, VD=4.5 mmand AP=O to the bregma, and into the right cortex at LM=2.5 mm, VD=2 mmand AP=O mm. Without wishing to be bound to any particular theory, thisposition approximates the ischemic boundary zone. Three microliters ofspheres were initially injected into the striatum and 2 ml into thecortex over a 10-minute period in each spot. The needle was retained inthe cortex for an additional 5 minute interval to avoid bone marrowreflux from the injected areas to the brain surface. After injection,bone wax (W810, Ethicon) was placed on the skull to prevent the leakageof the solution. Rats were sacrificed at 14 days after MCAo.

Traumatic Brain Injury (TBI)

BrdU prelabeled MSCs and neurospheres were mixed and cultured in flasksfor 7 days. At 4 days after TBI rats (n=4) were anesthetized withchloride hydrate and placed onto the stereotactic frame, and thenexposed to the previous injured area. A pipette with a glass tip (0.5 mmof diameter) containing 15 prepared mixed NMCspheres (diameter of 0.25mm) in 20 UL PBS was fixed onto the stereotactic frame. The tip of theneedle was inserted at the central site of the injured area, 2.5 mm awayfrom brain surface. Spheres were injected into the brain over 5 minutes,and then kept for an additional 5 minute interval to avoid reflux. Inboth sets of experiments (stroke and TBI) functional outcomemeasurements were measured using the rotarod and adhesive removal tests.

Results

Functional benefit in both stroke and TBI was evident in rats treatedwith NMCspheres. These data indicate that NMCspheres can be employed forthe treatment of stroke and brain injury. This composite, is a newmaterial with potential for the treatment of CNS injury andneurodegeneration.

Example 8 Description of Novel Medium (With and Without Growth Factors)Employed for the Culturing of MSCs for the Treatment of Neural Injuryand Neurodegeneration

Primary bone marrow cells were obtained at 48 hours after treating adultWistar rats with 5-fluorouracil (5-FU, 150 mg/kg) and cultured in theIscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetalbovine serum (FBS) and stem cell factor (100 ng/ml). Adherent MSCs wereresuspended in fresh IMDM with nerve growth factor (NGF, 200 ng/ml),brain derived neurotrophic factor (BDNF, 100 ng/ml) and epidermal growthfactor (EGF, 20 ng/ml) up to one month. Control MSCs were cultured inthe IMDM without neural growth factors. Antibodies against neuronalnuclei (NeuN), microtubule associated protein-2 (MAP-2) and glialfibrillary acidic protein (GFAP) were used for immunocytochemicalidentification of cultured cells.

The data indicates that cells derived from adult bone marrow stem andprogenitor cells can grow in large quantities in culture and expressproteins characteristic of neurons and astrocytes. Neurotrophic growthfactors enhance the neural expression of cells derived from bone marrowcells in vitro. Immunocytochemical staining shows that control MSCscultured without neurotrophic growth factors expressed the neuronalmarker, NeuN (˜1%, FIG. 1A) and the astrocytic marker, GFAP (˜3%, FIG.1B) at a baseline level. However, MSCs treated with neurotrophic growthfactors (i.e., NGF) express NeuN (˜3%, FIG. 1C) and GFAP (˜30%, FIG. 1D)at an elevated level.

Bromodeoxyuridine, which is incorporated into dividing cells, andidentifies newly formed DNA, was added to the medium at 72 hours beforetransplantation. Using immunoperoxidase with 3,3′-diaminobenzidine (DAB,brown) and counter staining by hematoxylin, bone marrow cells areidentified by the antibody against BrdU. The number of MSCs labeled withBrdU was observed to be ˜90% in vitro.

Discussion

The data demonstrate that cultured adult bone marrow cells, particularlymarrow stromal cells (MSCs), survive and differentiate into parenchymallike cells in the adult rodent brains after ischemia, brain and spinalcord trauma, and Parkinson's disease, and that bone marrow promotesprominent proliferation, differentiation and migration of ventricularzone/subventricular zone (VZ/SVZ) NSCs.

Pluripotent bone marrow cells become glia in normal rat brain (Azizi etal., 1998), and facilitate cell proliferation and cell-specificdifferentiation after MCAo. The bone marrow transplantation experimentrequires a sensitive means of monitoring the fate of the bone marrowcells. Help came from the bone marrow cells carrying tracers andmarkers, such as BrdU, CD34, nestin, PCNA. Pluripotent hematopoieticstem cells and mesenchymal stem cells from the adult bone marrow exposedto the new ischemic microenvironment after MCAo are triggered toproliferate and differentiate into neuronal (MAP-2, NeuN) and glial cell(GFAP) phenotypes. Fresh bone marrow or stroma humoral factors are alsoa source of differentiating factors and provides the chemotaticmicroenvironment to enhance the proliferation, migration anddifferentiation of neural stem cells from VZ/SVZ.

The VZ/SVZ of the mammalian forebrain is a region of germinal matricesthat develops late in gestation, enlarges, and then diminishes in size,but persists in a vestigial form throughout life (Gage 1998). In thenormal adult brain, the absence of forebrain neuronal productionreflects not a lack of appropriate neural stem cells, but rather a tonicinhibition and/or a lack of postmitotic trophic and migratory support.Although the signals that trigger the quiescent central nervous system(CNS) stem cells within the normal VZ/SVZ to enter the cell cycle haveyet to be resolved, the data show that a lesioned CNS is a differentenvironment than an intact CNS and markedly alters the terminaldifferentiated phenotype of the neural stem cells. Importantly, theVZ/SVZ in the adult forebrain is not a passive ischemia-threatened zone,located far from the ischemic areas (FIGS. 3F-H), but is an activetissue providing cells to reconstruct brain. VZ/SVZ cells proliferateand differentiate into neuronal and glial phenotypes after MCAo. Thesurvival of neurons arising from adult NSCs is dictated by both theavailability of a permissive pathway for migration and the environmentinto which migration occurs. New neurons depart the VZ/SVZ to enter thebrain parenchyma via radial guide fibers, which emanate from cell bodiesin the ventricular ependyma in adult rat (FIGS. 2K-L), and provide apermissive pathway for migration as found during development (Rakic1972). Mitosis within the graft and VZ/SVZ shows that ischemic injuredbrain together with the transplanted cells reverts to an early stage ofdevelopment to promote repair. The data are consistent with theobservation that adult brain can form new neurons (Gage 1998).

Example 9 Effects of Bone Marrow Stromal Cells Injected via DifferentRoutes (Intraarterial Versus Intravenous) After Stroke in Rats

The data disclosed herein demonstrate the beneficial effects ofadministering rat bone marrow stromal cell (rBMSC) to rats that haveundergone stroke using intraarterial (IA) or intravenous (IV) deliverysystems. In the present experiments, comparative effects of rBMSCsinjected via IA or IV on neurological function, neurogenesis, andangiogenesis in ischemic rats were analyzed. Young adult rats weresubjected into middle cerebral artery occlusion (MCAo) for two hours.After 24 hours, 2×10⁶ rBMSCs or phosphate buffered saline (PBS) wereinfused into the carotid artery or the tail vein of the rat. The ratswere sacrificed at day one (n=6), day seven (n=6), day fourteen (n=6)and day twenty eight (n=12) after cell injection, (half mammals for IAand half for IV at every time point); whereas the control mammals (n=6for IA and n=6 for IV) were sacrificed at day twenty eight after PBSinjection. Behavioral tests (an adhesive-removal test and a modifiedNeurological Severity Score, mNSS) were performed at 1, 7, 14, 21 and 28day after MCAo. BrdU immunohistochemistry was used to evaluateendogenous neurogenesis in both the subventricular zone (SVZ) and thesubgranular zone (SGZ). Immunohistochemistry was employed to measureangiogenesis in the ischemic boundary zone (IBZ). Significant (P<0.05)recovery of the adhesive-removal test and mNSS were found in both IA andIV groups as early as day seven and persisted to at least day twentyeight after cell injections compared with control mammals that did notreceive injection of rBMSCs. Immunohistochemistry results showed thatthe number of BrdU positive cells in the SVZ and SGZ significantlyincreased (P<0.05) during 7-14 day after stroke and returned to baselineat day twenty eight. Quantitative analysis using immunohistochemisterytechniques indicated that angiogenesis was significantly enhanced(P<0.05) by the rBMSC administration and persisted for at least daytwenty eight in the IBZ after the onset of stroke. It was observed thatno significant difference between IA and IV groups in all the aboveexaminations was detected at this cell dose. Based on the presentdisclosure, rBMSCs delivered to the ischemic brain through bothintracarotid and intravenous routes provide therapeutic benefits afterstroke.

Example 10 Correlated Expression of MT1-MMP and IGF-1 Genes withNeurological Recovery in Ischemic Rats Treated with Human Marrow StromalCells

The data disclosed herein demonstrate that human bone marrow stromalcells (hMSCs) can enhance neurogenesis and promote neural stem cellproliferation and migration following administering into an ischemicrat. Without wishing to be bound to any particular theory, it isbelieved that the neurological recovery in an ischemic rats using hMSCis determined by enhanced gene expression in the ischemic area inducedby hMSCs. To elucidate the molecular mechanisms underlying the hMSCeffect on stroke, differently expressed genes in the treated anduntreated ischemic brain tissue were identified. Rats were subjected topermanent occlusion of the right middle cerebral artery (MCAo) alone(n=18) and were injected intravenously with 3×10⁶ hMSCs (n=18) at dayone after MCAo. Functional outcome was measured at 0, 1 and 7 day afterMCAo by a modified Neurological Severity Score, mNSS. A number of genesincluding, but not limited to, Membrane-Type 1 matrix metalloproteinase(MT1-MMP), insulin-like growth factor (IGF-1) and its receptor IGF-1R inthe ischemic boundary zone (IBZ) were tested using RT-PCR at day 0, 2and 7 after MCAo in rats treated with hMSC, compared with control ratsnot receiving injection of hMSCs. RT-PCR analysis demonstrated asignificant increase of IGF-1 and MT1-MMP mRNA at day two after MCAowhich was observed to persist for at least day seven. IGF-1 mRNA levelsin the IBZ of rats receiving injection of hMSC at day two and day sevenafter MCAo were significantly increased compared with that of non hMSCtreated mammals. MT1-MMP mRNA levels in hMSC treated rats exhibited nodistinct difference from that of the non treated mammal at day two, butthere was a significant increase in MT1-MMP mRNA at day seven in hMSCtreated rates as compared with non treated mammals. It was also observedthat there was no significant difference in IGF1-R mRNA expression amongthe normal, ischemic and hMSC treated rats at any time points tested.These data indicate that hMSC treatment can achieve a neurorestorativeeffect through multiple mechanisms in the IBZ. Elevated IGF-1, in thepresence of abundant IGF-1 receptor, can promote neuron survival andregeneration during the hMSC treatment of these ischemic rats. MT-1 MMP,an important membrane-bound MMP, has been suggested to play a centralrole in mediating cell surface focal proteolysis pathways. IGF-1 hasbeen demonstrated to up-regulate MT1-MMP expression. The presentdisclosure demonstrates that MT1-MMP may be functionally linked toneuronal survival and regeneration mediated by IGF-1 in the IBZ.

Example 11 Treatment of Stroke in Rats with Human Bone Marrow StromalCells

The data disclosed herein addresses whether treatment of stroke in ratsusing xenogeneic human bone marrow stromal cells (hMSCs) necessitatesthe use of an immunosuppressive agent, for example, cyclosporin A (CsA),and whether CsA affects the neurological response to stroke andtreatment with hMSCs in rats. hMSCs were obtained from three healthyhuman donors. Adult Wistar rats were subjected to 2 hours of middlecerebral artery occlusion (MCAo). Four groups of ischemic rats (n=6, pergroup) were subjected to: 1) MCAo alone without treatment; 2) 15 mg/kgCsA by gastric feeding daily beginning at one day after MCAo for 27days; 3) tail intravenous injection of 3×10⁶ hMSCs at one day afterMCAo; and 4) co-treatment with hMSCs and CsA after MCAo. Functionaloutcome was measured by using an adhesive-removal patch test and amodified Neurological Severity Score (mNSS) before stroke and at 1, 7,14, 21 and 28 day after stroke. A human-specific antibody (Mab1281)against cellular nuclei was used to identify hMSCs within the braintissue. Since unwanted activation of T-lymphocytes promotes graftrejection, human graft-versus-rat host cytotoxic T lymphocyte (CTL)response was measured using a ⁵¹Cr assay to determine the lytic effect.It was observed that no stroke rats died after hMSC injection into therats. Significant functional recovery of adhesive-removal (p<0.05) at14, 21 and 28 day, and mNSS (p<0.05) at 21 and 28 day was found in ratstreated with hMSCs (Group 3 and 4), compared to control ischemia rats,which did not receive hMSC injection (Group 1 and 2). Few mAb1281positive cells (approximately 500-3,000 positive cells per brain) weredetected in recipient rats in Group 3 (1,283±592) and Group 4(1,431±727); however, no significant difference was observed betweenthese groups. It was also observed that CsA did not have an apparenteffect on neurological functional recovery after stroke with or withouthMSC treatment. There was no evidence of hMSC induction of CTL responseboth in vitro and in vivo. The data indicate that there is no apparentcomplicating immune response that obscures the therapeutic benefit ofhMSC treatment in stroke tats. Thus, CsA immunosuppression is not neededas an adjunctive therapy when administering hMSCs to a rat.

Example 12 Allogeneic Rat Marrow Stromal Cells Promote Brain RemodelingWithout Immunologic Sensitization in Stroked Rats

The present disclosure addresses the effects of allogeneic (allo-) andsyngeneic (syn-) rat bone marrow stromal cell (rBMSC) for the treatmentof stroke with respect to functional outcome based on immune reaction,glial scar formation and glial-axonal architecture. Female Wistar rats(n=25) were subjected to middle cerebral artery occlusion (MCAo) for twohours. At 24 hour after MCAo, rats were injected intravenously withphosphate buffered saline (PBS, n=8), syn-Wistar strain rBMSCs(3×10⁶/rat, n=8), or allo-ACI strain rBMSCs (3×10⁶/rat, n=9).Neurological functional recovery was performed using the NeurologicalSeverity Score, adhesive-removal patch and Corner tests. Rats weresacrificed at day twenty eight after treatment, and were bled todetermine antibody titers to rBMSCs. Lymphocytes collected frommesenteric and cervical lymph nodes were cultured with irradiated syn-or allo-spleen cells to determine T cell proliferative responses againstdonor alloantigens using the Mixed Lymphocyte Reaction assay. Antibodytiters to rBMSCs were determined by a flow cytometry method. In situhybridization and double immunostaining techniques were employed formale Y-chromosome⁺ bearing rBMSC and brain cell type identification.Significant functional recovery (p<0.05) was found in both groupstreated with rBMSCs (syn- or allo-) compared to PBS controls, but nodifference was detected between syn- and allo-rBMSC treated rats.Similar numbers of Y-chromosome⁺ cells were detected in the syn- andallo-rat brains at twenty eight days after treatment. Astrocyteproliferation was prominent (BrdU+-GFAP+, hyperplasia) in the ischemicbrain and exceeded cell proliferations in other cells. It was observedthat the thickness of the scar wall decreased (p<0.05), and the axonaldensity as measured by Bielshowsky silver staining increased (p<0.05) inareas where reactive astrocytes were present (GFAP+ hypertrophy) in thescar boundary zone (SBZ) and in the subventricular zone (SVZ) of therBMSC treated rats, compared with the non-treated rats. Moreover, axonalprojections exhibited an overall orientation parallel to elongatedprocesses of reactive astrocytes and toward lesion areas in the SBZ andSVZ of the rBMSC treated rats, suggesting that rBMSCs may enhancereactive astrocyte-related axonal repair in adult brain. No evidence ofT cell priming or humoral antibody production rBMSC was observed inrecipient mammals after treatment with allogeneic ACI-rBMSCs. Based onthe present disclosure, both syn- and allo-rBMSC treatment of stroke inrats improved neurological recovery and enhanced brain remodeling withno indication of immunologic sensitization.

Example 13 BMSC Confer Post-Ischemic Protection via Increased Akt andErk and Growth Factor Production Within Neighboring Astrocytes

It has been demonstrated that treatment of stroke using bone marrowstromal cells (BMSCs) significantly improves functional outcome, andreduces apoptosis in the brain. Astrocytes have been shown to be thefirst cells to suffer ischemic insult among all types of neural cells.The present disclosure provides insight on the interaction between BMSCsand astrocytes after ischemic insult. The data disclosed here addressedthe effect of rat BMSCs (rBMSCs) on post-ischemia induced apoptosis andcell death of astrocytes, as well as the mechanisms of these effectsusing an in vitro ischemic model. After a four hour ischemic incubationin an anaerobic chamber, astrocytes were co-cultured with rBMSCs innon-ischemic conditions (as post-ischemic incubation) for an additionalfour hours. Astrocytes cultured without the presence of rBMSCs (controlgroup) depicted evident morphological and biochemical apoptoticfeatures. A large number of condensed nuclei were observed, and manycells appeared as dark detached spheres or oval-shaped bodies. A cellviability assay demonstrated that about 35% of the astrocytes were notviable. However the introduction of rBMSCs remarkably reduced theapoptosis and cell death (to about 1.5%, P<0.01) in astrocytes, and mostof the astrocytes appeared as a confluent cobblestone layer.BrdU-immunostaining revealed a higher proliferation rate in theco-cultured astrocyte group compared to the control group. Western blotanalysis and real-time quantitative PCR demonstrated that rBMSCsdrastically increased Erk1 and Akt at both protein and RNA level inpost-ischemic astrocytes. Additionally, Western blot analysis alsorevealed that co-culturing astrocytes with rBMSCs upregulated thephosphorylation of Erk1 and Akt in astrocytes. Astrocytes treated withMEK inhibitor (U0126) or PI3K inhibitor (LY29004) underwent significantapoptosis and cell death similar to the post-ischemic control group.Co-culturing astrocytes with rBMSCs significantly (P<0.01) attenuatedthis U0126 and LY29004 mediated insult. Furthermore, real-time PCRdemonstrated that rBMSC co-culture increased RNA levels of bFGF, BDNF,and VEGF in astrocytes that had suffered ischemia. These resultsindicate that rBMSCs enhance the recovery of post-ischemia astrocytes bystimulating the activation of MEK/Akt and PI3K/Erk pathways inastrocytes, and increasing growth factor production by astrocytes.

Example 14 Treatment of Stroke in Rats with Human Marrow Stromal CellsDecreases Axonal Loss and Demyelination

Axonal loss and demyelination are frequently observed in ischemiccerebrovascular diseases and contribute to neurological functionalimpairment. It has been demonstrated that human marrow stromal cells(hBMSCs) improved neurological functional recovery in ischemic rats. Thepresent disclosure, addresses the effect of hBMSCs on axonal fibers inischemic brain. Rats were subjected to permanent middle cerebral arteryocclusion (MCAo) and injected intravenously with 3×10⁶ hBMSCs orphosphate buffered saline (PBS) (n=6 per group) at one day after MCAo,and sacrificed at fourteen days after MCAo. Axon and myelin damage wasexamined using Bielshowsky and Luxol fast blue double staining,respectively, in the MCAo rats receiving hBMSC or PBS treatment. Nervefiber damage was found in the white matter (WM) of the striatum (ST) andcorpus callosum (CC) of the ipsilateral hemisphere after MCAo with PBStreatment, and involved both axonal and myelinated components.Demyelination was more severe than axon loss (10.4±2.3% vs 3.7±0.7%),indicating that the myelin is more susceptible to ischemia than theaxon. The surviving WM area within the ipsilateral CC significantlyincreased compared to the corresponding area of the contralateral CC.Enhanced density of axons was observed in the WM bundles in the ST andWM in the CC of the ischemic boundary zone, indicating that aself-neurorestorative mechanism was initiated. It was observed that nosignificant change in the contralateral CC area among normal, PBS andhBMSC treatment groups. hBMSC treated MCAo mammals demonstratedsignificantly reduced areas of demyelination (3.7±0.2% vs 10.4±2.3%) andaxon loss (1.8±0.4% vs 3.7±0.7%), in the ipsilateral ST when compared toPBS treated controls. It was observed that the morphologically intactareas of the ipsilateral CC were significantly increased (19.8±4.5% vs11±6.7%); and the density of axons were enhanced (27.5±6.3% vs19.8±5.6%) in the ST and CC of the ischemic boundary zone in the hBMSCtreated rats compared with the PBS controls. The present disclosuresuggests that axonal and myelination remodeling may contribute toimproved functional recovery after treatment of stroke with hBMSCs.

In summary, the data indicate that intracerebral and intravascular bonemarrow transplantation after stroke neural injury and Parkinson'sdisease significantly improves functional recovery. Transplantation alsoenhances the proliferation and differentiation of exogenous bone marrowstem cells and endogenous NSCs. Bone marrow aspirations and biopsieshave been employed in the diagnosis and treatment of clinical diseases.Bone marrow transplantation provides a new avenue to induce plasticityof the injured brain and spinal cord and provides a therapeutic strategyfor treatment of neural injury and neurodegeneration.

In addition, a new substance is identified herein, a composite of MSCsand neurospheres, which when transplanted into brain after stroke ortrauma, improves functional recovery.

Example 15 Bone Marrow Stromal Cells Reduce Axonal Loss in theExperimental Autoimmune Encephalomyelitis (EAE) Mice

The following experiments were designed to investigate the effects oftransplantating human bone marrow stromal cells (hBMSCs) or otherwisehMSCs in an experimental model of multiple sclerosis (MS). Such anexperimental model involves assessing remitting-relapsing and axonalloss in experimental autoimmune encephalomyelitis (EAE) mice.

The present disclosure demonstrates that hBMSCs were able to reduceaxonal loss in an MS model. Briefly, EAE was induced in SJL/J mice(n=63) by injection with proteolipid protein (PLP). Mice were injectedintravenously with hBMSCs (n=26) or PBS (n=37) on the day of clinicalonset and neurological function was measured daily (score 0-5) until 45weeks after onset. Mice were sacrificed at week 1, 10, 20, 35 and 45.Double staining for Luxol fast blue and Bielshowsky was used to identifymyelin and axons, respectively. Immunohistochemistry was performed tomeasure the expression of nerve growth factor (NGF) and MAB1281, amarker of hBMSCs. hBMSC treatment significantly reduced the mortality ofEAE mice, and significantly improved functional recovery in EAE micecompared to PBS treatment. Axonal density in the EAE striatum and corpuscallosum was significantly increased in the hBMSC treatment groupcompared with that of the PBS treatment group. NGF⁺ cells significantlyincreased in the hBMSC treated mice compared to PBS controls at 1, 10,20, 35 and 45 weeks. Most of the NGF⁺ cells were identified as brainparenchymal cells. Less than 5% of MAB1281⁺ cells co-localized withNG2⁺, a marker of oligodendrocyte progenitor cells. About 10% ofMAB1281⁺ cells co-localized with GFAP and MAP-2, a marker of astrocytesand of neurons, respectively. It was observed that hBMSCs improvedfunctional recovery and therefore provides a therapy aimed at axonalprotection in EAE mice, in which NGF plays an important role.

The Materials and Methods used in the experiments presented in thisExample are now described.

Cell Culture

hBMSCs were isolated, grown and tested, using methods adopted from Zhanget al. (2005, Exp. Neurol. 195:16-26). Briefly, bone marrow was obtainedfrom adult human donors and the nucleated cell fraction was culturedwith Dulbecco's Modified Eagle Medium-low glucose media and 10% fetalbovine serum. The adherent cells were harvested, passaged andcryopreserved in appropriate dose related aliquots in Plasma-Lytecontaining human serum albumin and dimethyl sulfoxide. The cells aretested for purity at the end of each passage by flow cytometry. TheBMSCs were positive for MHC class I, CD29, CD90, CD105, CD13, CD44,CD63, CD73 and CD166. The cells were negative for MHC class II, CD45,CD14 and CD34.

EAE Induction and Animal Groups

Myelin proteolipid protein (PLP) (p139-151; HSLGKWLGHPDKF, SEQ ID NO:1;SynPep Corporation, Dublin, Calif.) was used for immunization. Thepurity of the peptide was greater than 95% as measured by HighPerformance Liquid Chromatography. EAE was induced in female SJL/J mice(8-10 week old, Jackson Laboratory, Bar Harbor, Me.) by subcutaneousinjection with 25 ug PLP dissolved in 50 ul complete Freund's adjuvant(CFA) (Difco Laboratories, Livonia, Mich.). On the day of immunizationand 48 hours post immunization, 200 ng pertussis toxin (PT) (ListBiological laboratories, Inc. Campbell, Calif.) in 0.2 ml phosphatebuffered saline (PBS) was injected into the mouse tail vein (Youssef etal., 2002, Nature 420:78-84; Zhang et al., 2005, Exp. Neurol.195:16-26). Mice were randomly divided into: 1) hBMSC treatment group(n=26): hBMSCs (2×10⁶ per mouse) were administered intravenously in 1(one) ml total fluid volume PBS on the day of clinical symptom onset(score≧1); and 2) PBS treatment group (n=37): PBS (1 ml) was injectedinto the tail vein of the EAE mice on the day of clinical symptom onsetas EAE controls. An additional control normal group (n=6) consisted ofmice without immunization.

Neurological Functional Measurement

Mice in the hBMSC treatment group and PBS treatment group were scoreddaily for clinical symptoms of EAE, as follows: 0, healthy; 1, loss oftail tone; 2, ataxia and/or paresis of hindlimbs; 3, paralysis ofhindlimbs and/or paresis of forelimbs; 4, tetraparalysis; 5, moribund ordead (Pluchino et al., 2003, Nature 422:688-694). Neurological functionsof EAE were tested in mice treated with hBMSCs or PBS daily until 45weeks after clinical symptom onset.

Histopathology and Immunohistochemistry

EAE mice treated with PBS or hBMSCs were euthanized at 1, 10, 20, 35 and45 weeks after clinical symptom onset. Brain tissue (ranging from bregma+1.18 mm to bregma −1.82 mm) were fixed in 4% of paraformaldehyde anddivided into 4 serial sections per mouse. These tissue blocks wereembedded in paraffin and cut into 6 μm thick coronal slides.

Double staining for Luxol fast blue and Bielshowsky was used todemonstrate myelin and axons, respectively (Karnezis et al., 2004, Nat.Neurosci. 7:736-744; Pluchino et al., 2003, Nature 422:688-694; Furlanet al., 2001, J. Immunol. 167:1821-1829). After staining, nucleiappeared colorless; myelin turquoise and axons appeared black on a palegrey/blue background.

To identify the expression of NGF, a rabbit polyclonal antibody againstNGF (1:300, Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) was used.To identify the fate of injected hBMSCs in the CNS of EAE mice, slideswere treated with a monoclonal antibody specific to human nuclei(MAB1281; 1:500, Chemicon, Temecula, Calif.). Double immunofluorescencelabeling was performed to identify the relationship of NGF with neuralcells and hBMSC with neural cells. Antibodies against MAP-2 or NeuN(markers of neurons), glial fibrillary acidic protein (GFAP, a markerfor astrocytes) and NG2 (a marker for progenitor oligodendrocyte cell)were used to identify parenchymal cells. Negative control slides foreach animal received identical preparations for immunostaining, exceptfor the fact that primary antibodies were omitted.

Quantification and Statistical Analysis

Neurological functional tests and tissue slides were evaluated by ablinded examiner to the treatment status of each animal. Mice weremonitored for mortality up to 45 weeks (i.e., if the mice were notsacrificed earlier than 45 weeks). Mortality rates were compared betweenthe hBMSC treated and PBS treated groups using the log-rank survivalanalysis with Kaplan-Meier curves plotted for survival rates over time.

The functional score, ranging from 1 to 5, was measured on the daybefore the treatment and daily during and after the treatment up to 45weeks after EAE. Subgroups of mice were sacrificed at week 1 (n=7 ineach group), week 10 (n=7 in the PBS group; n=4 in the hBMSC group),week 20 (n=4 in each group), week 35 (n=6 in the PBS group, n=4 in thehBMSC group) and week 45 (n=4 in each group) for measurement ofmorphological changes. In the case where a mouse died prior to anupcoming sacrificed time, the functional score 5 was given for thatsacrifice time.

Normality of the functional recovery score was evaluated usingGeneralized Estimating Equations (GEE) on the ranked data. Withoutwishing to be bound by any particular theory, GEE was chosen because ithas fewer restrictions on the data distribution. Analysis of variancefor repeated measures including the independent factor of the treatmentand dependent factor of the time was also employed. Analyses wereperformed on scores measured from week 1 to week 45. The analysis begantesting for the treatment by time interaction, followed by testing themain effect of hBMSC or time, if no interaction was observed at the 0.05level. A subgroup analysis of hBMSC effect at each time point wasconducted, if an interaction or time effect was detected. The hBMSC bytime interaction indicated that the effect of hBMSC on the functionalrecovery depended on the time after EAE. Functional outcome was reportedas mean±SD per time point for data illustration.

Axonal loss in the white matter of the corpus callosum and striatum inthe EAE brain was assessed. The axonal density was counted on an averageof four brain sections (ranging from bregma +1.18 mm to bregma −1.82 mm)per mouse (at 40× magnification). Data were obtained using a 3-CCD colorvideo camera (Sony DXC-970 MD) and interfaced with ImageJ imageprocessing program (National Institute of Mental Health, Bethesda, Md.).The axonal density was presented as a proportional area. To measureimmunoreactive cells, numbers of NGF⁺ and MAB1281⁺ cells were counted onan average of 4 brain sections (ranging from bregma +1.18 mm to bregma−1.82 mm) per mouse (at 40× magnification), using a 3-CCD color videocamera (Sony DXC-970 MD) interfaced with the Micro Computer ImagingDevice (MCID) analysis system (Imaging Research Inc. St. Catharines,Ontario, Canada). The density of immunoreactive cells was calculated bydividing the number of counted cells by the scan area, presented asnumbers per mm². Data was presented as mean±SD. Significance between thetwo groups was examined using a t-test. A value of p<0.05 was consideredsignificant.

The results of the experiments presented in this Example are nowdescribed.

hBMSC Treatment Improves Survival Rate and Neurological FunctionalRecovery in EAE Mice

Without wishing to be bound by any particular theory, since MS is achronic disease course, the mortality and function of the mice weremeasured up to 45 weeks after clinical onset. It was observed that micein the hBMSC treated group had significantly higher survival rates ascompared to the PBS treated group. A total of 63 EAE mice were employedin the study. Survival rates for hBMSC treated mice at weeks 10, 20, 35,and 45 were significantly higher than those in the PBS group (p<0.01)(FIG. 12A).

Experiments were designed to evaluate whether the administration ofBMSCs on the day of clinical onset was an effective treatment. It wasobserved that there were several remitting-relapsing courses of diseasewithin 45 weeks after clinical symptom onset (FIG. 12B). Therelationship between hBMSC treatment and time was significant (p<0.05)and therefore, pair-wise comparisons at each time point were conductedwith the mean and SD of the functional scores. Functional scores weresignificantly lower among mice treated with hBMSCs compared with PBStreated mice as early as 1 week up to 45 weeks. Before week 20,functional scores were significantly lower in the hBMSC group comparedwith the PBS group at 70% time points, after week 20, there weresignificant difference between 2 groups at 20% time points. Thesignificance of hBMSCs effects were sustained to 45 weeks (p<0.05) (FIG.12B).

hBMSC Treatment Increases Axonal Density in the White Matter of the EAEBrain

Due to the effective neurological functional benefit of hBMSC treatment,experiments were designed to address whether BMSC treatment affectsaxonal loss. The proportional area of axonal loss was significantlyreduced in the striatum (FIGS. 13A and 13B) and corpus callosum (FIGS.13C and 13D) of the hBMSC treatment group compared with that of the PBStreatment group at 20, 35 and 45 weeks after clinical onset. These datademonstrate that the long term functional effect of BMSCs is associatedwith the reduction of axonal loss in the EAE brain.

Administration of hBMSCs Increases NGF Expression in the CNS of EAE Mice

Without wishing to be bound by any particular theory, it is believedthat BMSC treatment reduces axonal loss and improves neurologicaloutcome by augmenting expression of NGF in parenchymal cells. Thefollowing experiments were designed to measure NGF cell expression inthe white matter of the striatum and corpus callosum. It was observedthat NGF was present in the normal brain tissue of mice. After onset ofEAE, cellular expression of NGF significantly decreased in the brainduring the acute and chronic phase of EAE. hBMSC treatment significantlyincreased the number of NGF reactive cells in the brain at 1, 10, 20, 35and 45 weeks compared with the PBS treatment (FIGS. 14A and 14B).Furthermore, double staining shows that hBMSCs stimulated the brainparenchymal cells to express NGF. Approximately, 50-70% of NGF⁺ cellsco-localized with NeuN⁺ cells (FIG. 14C).

hBMSCs are Present in the EAE Brain

MAB1281⁺ cells were present in the CNS from as early as 1 week up to 45weeks following hBMSC transplantation. Most of the cells were located inthe striatum and the corpus callosum. The number of MAB1281⁺ cellssignificantly increased at 10, 20, 35 and 45 weeks compared to theMAB1281⁺ cells at 1 week (FIG. 15). Double staining revealed that lessthan about 5% of MAB1281⁺ cells co-localized with NG2⁺ cells and about10% of MAB1281⁺ cells co-localized with MAP-2⁺ cells and GFAP⁺ cells,respectively, (FIG. 16). These data demonstrated that the therapeuticeffect of BMSCs on EAE was not a result of cell replacement.

BMSCs for the Treatment of Degenerative Diseases of the CNS

The results presented herein demonstrate that transplantation of hBMSCsat the day of clinical EAE symptom onset improved survival rates andreduced disease severity, having a statistical significance from 1 weekup to 45 weeks after disease compared with PBS treatment.

Since MS is an immune-mediated demyelinating and degenerative disease ofthe CNS, with lesions predominantly occurring in the CNS white matter,it is believed that the first step in combating MS is to suppress theimmune onslaught. However, these strategies alone are insufficient fortreating the chronic progressive disability that is the ultimate outcomeof the disease (Chitnis et al., 2005, Curr. Drug Targets Immune Endocr.Metabol. Disord. 5:11-26).

The data presented herein revealed that few MAB1281⁺ cells co-localizedwith neural cell markers. Although the injected hBMSCs express braincell phenotypic proteins, the data does not indicate truedifferentiation and neuronal or glial cell function. Thus, it isbelieved that the beneficial outcome from hBMSC treatment is not aresult of a cell replacement therapy. Rather, it is believed that BMSCssecrete a series of growth factors and induce expression of growthfactors within the parenchymal cells.

In the present study, it was observed that NGF expression increasedafter hBMSC treatment. NGF stimulates axonal repair (Walsh et al., 1999,J. Neuroscience 19:4155-4168; Jones et al., 2003, J. Neurosci.23:9276-9288) and induces axon growth (Zhou et al., 2004, Neuron42:897-912). Moreover, axon repair is present not only in acute, butalso in chronic CNS injury (Grill et al., 1997, Exp. Neurol.148:444-452). NGF also enhances the survival of differentiatedoligodendrocytes (Cohen et al., 1996, J. Neurosci. 16:6433-6442), andstimulates oligodendrocyte growth and/or differentiation (Aloe et al.,1998, Arch. Ital. Biol. 136:247-256). In addition to its neurotrophiceffect, NGF exhibits immunomodulatory effects (Aloe et al., 1997,Allergy 52:883-894; Flugel et al., 2001, Eur. J. Immunol. 31:11-22;Fainzilber et al., 2002, J. Neurosci. 3:1029-1034; Aloe et al., 2004,Ann. Ist Super Sanita. 40:89-99), such as suppression of MHC IIinducibility in microglia and stimulation of memory B cells and Th2responses (Bracci-Laudiero et al., 2002, J. Neuroimmunol. 123:58-65;Bonini et al., 2003, Int. Arch. Allergy Immunol. 131:80-84;Bracci-Laudiero et al., 2005, Blood 106:3507-3514; Stampachiacchiere etal., 2005, J. Neuroimmunol. 169:20-30). It has also been found thatadministration of NGF dramatically reduced the number and size oflesions produced in EAE, upregulated the anti-inflammatory cytokineIL-10 in glial cells and suppressed interferon-γ expression byinfiltrating T-cells (Villoslada et al., 2002, J. Exp. Med.191:1799-1806). Anti-NGF treatment of rats resulted in more severe EAEpathology (Micera et al., 2000, J. Neuroimmunol. 104:116-123). Withoutwishing to be bound by any particulate theory, it is believed thatstimulation of NGF in the parenchymal cells by hBMSCs contributes to thereduction of axonal injury and improvement of neurological outcome.

The results presented herein demonstrate that MAB1281+ cellssignificantly increased at 10, 20, 35 and 45 weeks compared to theMAB1281+ cells at 1 week after hBMSC transplantation. It is believedthat the increased number of hBMSCs present after transplantationfacilitates functional recovery. It was observed that hBMSCs entered theCNS of EAE mice and contributed to the decreased mortality and improvedneurological functional recovery from as early as 1 week up to 45 weeksin the mice. The transplanted cells contributed to the reduced axonalloss in the EAE brain, and stimulated brain parenchymal cells to expressNGF, which is believed to provide both neurotrophic and immunomodulatoryeffects. The results presented herein demonstrate that BMSC treatment isuseful for therapy of autoimmune demyelinating disorders.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of treating a mammal having an autoimmune demyelinatingdisease/disorder, the method comprising isolating a stromal cell from abone marrow sample, and administering said stromal cell to said mammal,wherein the presence of said stromal cell in the CNS of the mammaleffects treatment of said disease/disorder, further wherein the presenceof said stromal cell in the CNS of the mammal reduces axonal loss. 2.The method of claim 1, wherein said stromal cell is selected from thegroup consisting of an autologous stromal cell, an allogenic stromalcell, a syngeneic stromal cell, and a xenogeneic stromal cell, withrespect to said mammal.
 3. The method of claim 1, wherein said mammal isa human.
 4. The method of claim 1, wherein said stromal cell is derivedfrom a human donor.
 5. The method of claim 1, wherein said autoimmunedemyelinating disease/disorder is selected from the group consisting ofa genetic disease, multiple sclerosis (MS), and a neurodegenerativedisease. 6.-8. (canceled)
 9. The method of claim 1, wherein said stromalcell administered to said CNS remains present or replicates in said CNS.10. The method of claim 1, wherein said stromal cell administered tosaid CNS does not result in a cell replacement therapy.
 11. The methodof claim 1, wherein said stromal cell administered to said CNS inducesexpression of a growth factor within neighboring cells. 12.-13.(canceled)
 14. The method of claim 1, wherein prior to administeringsaid stromal cell to said mammal, said cell is cultured in vitro for aperiod of time.
 15. The method of claim 1, wherein prior toadministering said stromal cell to said mammal, said stromal cell istransfected with an isolated nucleic acid encoding a therapeuticprotein, wherein when such protein is secreted by said stromal cell,said protein serves to effect treatment of said disease/disorder. 16.The method of claim 1, wherein said stromal cell is administered to saidmammal by a route selected from the group consisting of intreavascular,intracerebral, parenteral, intraperitoneal, intravenous, epidural,intraspinal, intrastemal, intra-articular, intra-synovial, intrathecal,intra-arterial, intracardiac, and intramuscular.
 17. The method of claim1, wherein said stromal cell is administered to said mammal at the siteof injury.
 18. The method of claim 1, wherein said stromal cell isadministered to said mammal at an adjacent site to the site of injury.19. (canceled)
 20. The method of claim 1, wherein said stromal cellpresent in the CNS activates the proliferation of neighboring cells. 21.The method of claim 1, wherein said stromal cell is administeredconcomitantly with a growth factor.
 22. The method of claim 1, whereinsaid stromal cell administered to said mammal prevents axonal fiber lossin the cells of the mammal.
 23. The method of claim 1, wherein saidstromal cell administered to said mammal prevents demyelination in thecells of the mammal.
 24. (canceled)
 25. The method of claim 1, whereinsaid stromal cell in the CNS of the mammal induces angiogenesis.
 26. Themethod of claim 1, wherein said stromal cell in the CNS of the mammalinduces neurogenesis.
 27. The method of claim 1, wherein said stromalcell in the CNS of the mammal induces synaptogenesis.